Sequences characteristic of hypoxia-regulated gene transcription

ABSTRACT

There are provided polynucleotides which are modulated by hypoxic conditions. The disclosure includes such genes and proteins as well as analogs, salts and functional derivatives of such proteins, and DNA encoding such analogs, and methods of use. Methods for treating the effects of stroke, hypoxia and/or ischemia by regulating such genes or proteins are also disclosed. The presence of hypoxia or a hypoxia-associated pathology may be diagnosed by screening for the presence of at least one polynucleotide having the nucleic acid sequence according to the present invention. Methods of regulating hypoxia associated pathologies are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part application of U.S.application Ser. No. 09/383,096, filed Aug. 27, 1999, which is acontinuation-in-part of U.S. application Ser. No. 09/138,109, filed Aug.21, 1998, and a conversion of U.S. provisional applications No.60/098,158, filed Aug. 27, 1998 and No. 60/132,684, filed May 5, 1999,the entire contents of all of which are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field

[0003] The present invention relates to the identification of genes thatare differentially expressed in hypoxia and use of the genes and geneproducts for diagnosis and therapeutic intervention. The inventionfurther relates to identification of polynucleotide sequences, and theirgene products, that are differentially expressed in hypoxia and the useof the sequences for diagnosis and probes.

[0004] 2. Background Art

[0005] The level of tissue oxygenation plays an important role in normaldevelopment as well as in pathologic processes such as ischemia. Tissueoxygenation plays a significant regulatory/inducer role in bothapoptosis and in angiogenesis (Bouck et al, 1996; Bunn et al, 1996; Doret al, 1997; Carmeliet et al, 1998). Apoptosis (see Duke et al, 1996 forreview) and growth arrest occur when cell growth and viability arereduced due to oxygen deprivation (hypoxia). Angiogenesis (i.e. bloodvessel growth, vascularization) is stimulated when hypo-oxygenated cellssecrete factors which stimulate proliferation and migration ofendothelial cells in an attempt to restore oxygen homeostasis (forreview see Hanahan et al, 1996).

[0006] Hypoxia plays a critical role in the selection of mutations thatcontribute to more severe tumorogenic phenotypes (Graeber et al, 1996).Identifying activated or inactivated genes and gene products in hypoxiaand ischemia is needed.

[0007] Ischemic disease pathologies involve a decrease in the bloodsupply to a bodily organ, tissue or body part generally caused byconstriction or obstruction of the blood vessels, as for exampleretinopathy, myocardial infarction and stroke. Therefore, apoptosisand/or angiogenesis as induced by the ischemic condition are alsoinvolved in these disease states. Neoangiogenesis is seen in some formsof retinopathy and in tumor growth. These processes are complex cascadesof events controlled by many different genes reacting to the variousstresses such as hypoxia.

[0008] Stroke is the third leading cause of death and disability indeveloped countries, affecting more than half a million Americans eachyear. Stroke is an acute neurologic injury occurring as a result of aninsult to the brain, thus interrupting its blood supply. Stroke inducesneuronal cell death, which leads to the clinical outcomes of patients'death or disability ranging from total paralysis to milder dysfunction.Cerebral ischemia is the most common type of stroke, which may lead toirreversible neuronal damage at the core of the ischemic focus, whereasneuronal dysfunction in the penumbra may be reversible. Cells in thepenumbra have an estimated time window for survival of up to 6 hours.The ability to intervene as soon as the patient is identified isessential for recovery. It is well established that ischemic tissuedamage is multifactorial and involves at least excitotoxicity, reactiveoxygen species, and inflammation—all leading to neuronal cell death.

[0009] Treatment strategies for stroke are aimed to induce rapidreperfusion and rescue of neurons in the penumbral area. Neuroprotectivedrugs are constantly being developed in an effort to rescue neurons inthe penumbra from dying. However, potential cerebroprotective agentsneed to counteract all the above-mentioned destructive mechanisms.Therefore, current therapy in stroke focuses primarily on prevention,minimizing subsequent worsening of the infarction, and decreasing edema.

[0010] The ability to monitor hypoxia-triggered activation of genes canprovide a tool to identify not immediately evident ischemia in apatient. Identification of hypoxia-regulated genes permits theutilization of gene therapy or direct use of gene products, oralternatively inactivation of target genes for therapeutic interventionin treating the diseases and pathologies associated with hypoxia,ischemia and tumor growth.

[0011] Induction of p53 in response to hypoxia and DNA damage and itsability to inhibit cell growth in response to common cellular stresses,is a major function associated with its role as a tumor suppressor gene(Lane, 1992). Proteins encoded by p53 target genes have been shown toregulate various processes controlling growth and viability of tumorcells, such as cell cycle progression and programmed cell death. Likep53, the growth arrest and DNA damage (GADD) genes are induced in cellsexposed to genotoxic stress. GADD genes were originally identified bysubtraction hybridization from a cDNA library constructed fromUV-irradiated Chinese hamster ovary cells (Forance et al, 1989). TheGADD genes code for a diverse range of proteins with a variety offunctions, including the suppression of DNA synthesis (Smith et al,1994), the inhibition of differentiation (Batchvarova et al, 1995) andthe induction of apoptosis (Takekawa et al, 1998). The response togenotoxic stress of some GADD genes is rapid but transient whereasothers respond more slowly (Fleming et al, 1998). Other stimuli, such asDNA damage or contact inhibition, also increase gene expression. Theregulation of these genes by stress is complex and appears to bemediated by multiple pathways. For example, ionizing radiation inducesthe transcription of GADD45, which inhibits proliferation and stimulatesDNA excision repair, through a p53-dependent mechanism (Hollander et al,1993). In contrast, UV irradiation increases GADD45 expression in theabsence of p53 binding directly to the GADD45 promoter (Zhan et al,1996). GADD45 mRNA levels are also increased during hypoxia, focalcerebral ischemia, and after exposure of cells to agents which elevatethe levels of the glucose-regulated proteins (Price et al, 1992,Schmidt-Kastner et al, 1998). In addition to its ability to inhibitproliferation and stimulate DNA repair, GADD45 can also induce apoptosiswhen overexpressed in cells in vitro (Takekawa et al, 1998).

[0012] Recently, a novel p53 target gene and member of the GADD family,PA26 was identified (Velasco-Miguel et al, 1999). PA26 encodes at leastthree transcript isoforms, of which two are differentially induced bygenotoxic stress in a p53-dependent manner. The function of PA26 isunclear.

SUMMARY OF THE INVENTION

[0013] The present invention provides purified, isolated and clonedpolynucleotides (nucleic acid sequences) associated withhypoxia-regulated activity and having sequences designated as any one ofSEQ ID NOs: 1-12, or having complementary or allelic variation sequencesthereto. The expression of these polynucleotides is modulated when cellsare subjected to neurotoxic stress. The present invention includes thepolynucleotides of SEQ ID NOs: 1-12, as well as the naturally-occurringfull-length RNAs and corresponding full-length cDNAs which include anyone of these sequences.

[0014] The invention is further directed to naturally-occurringpolynucleotides having at least 70% identity with any of thepolynucleotides which include any one of SEQ ID NOs: 1-12, or which arecapable of hybridizing under moderately stringent conditions to any ofsuch polynucleotides, and whose expression in naturally-occurring neuralcells is modulated when the cells are subjected to hypoxic stress.

[0015] The present invention is also directed to fragments having atleast 20 nucleotides of any of the polynucleotides of the presentinvention and to polynucleotide sequences complementary to any of suchpolynucleotides or fragments.

[0016] In a preferred embodiment, the isolated polynucleotide is astrand of a full-length cDNA.

[0017] The present invention is further directed to isolated proteinsencoded by any such full-length cDNA, as well as variants which have anamino acid sequence having at least 70% identity to such an isolatedprotein and retain the biological activity thereof, or biologicallyactive fragments of such protein or variant, as well as to salts orfunctional derivatives of any such protein, variant or biologicallyactive fragment.

[0018] The present invention is also directed to antibodies specific toany of the proteins, variants or fragments of the present invention andto any molecule which includes the antigen-binding portion of any suchantibody.

[0019] The present invention also comprehends antisense DNA of a lengthsufficient to prevent transcription and/or translation of a geneidentified in accordance with the present invention, as well asribozymes which specifically bind and cleave mRNA sequences identifiedin accordance with the present invention.

[0020] The invention also comprehends methods for screening drugs whichup-regulate or down-regulate a gene which is transcribed to an RNAcontaining a sequence of any of any of the polynucleotides of thepresent invention.

[0021] The present invention is additionally directed to pharmaceuticalcompositions which include the nucleic acids, proteins or polypeptidesin accordance with the present invention, along with pharmaceuticallyacceptable carriers or excipients.

[0022] In addition, the present invention is directed to knockout ortransgenic non-human animals, in which a gene identified by the presentinvention has been introduced or knocked out.

[0023] The present invention further provides a method of regulatingangiogenesis or apoptosis in a patient in need of such treatment byadministering to such patient a therapeutically effective amount of anantagonist of at least one protein as encoded by the nucleic acidsequences in accordance with the present invention.

[0024] Also provided is a diagnostic method for identifying genesmodulated by hypoxic conditions by detecting the presence of apolynucleotide having a nucleic acid sequence according to the presentinvention.

[0025] Also provided is a method of regulating hypoxia-associatedpathologies by administering an effective amount of at least oneantisense oligonucleotide against one of the nucleic acid sequences (SEQID NOs: 1-12) or their proteins. There is provided a method ofregulating hypoxia associated pathology by administering an effectiveamount of a protein encoded by the polynucleotides (SEQ ID NOs: 1-12) asactive ingredients in the pharmaceutically acceptable carrier.

[0026] Further, there are provided hypoxia response regulating genes.

[0027] Among the genes in accordance with the present invention is thenovel gene 95, which shares homology with the PA26 gene. The mRNA levelsof gene 95 are increased during hypoxia, regardless of the p53 status ofthe cells. In contrast, DNA damaging agents induce 95 expression in ap53-dependent manner. 95 is involved in regulation of cell survivalunder ischemia and hydrogen peroxide; however, it induces DNA damagedapoptosis. Conditioned medium from 95 overexpressing clones alsopossesses pro-apoptotic activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 shows sequence comparison of protein 95 (SEQ ID NO: 4) andPA26 (SEQ ID NO: 21).

[0029]FIG. 2 is a graph showing how 95 overexpression affects the growthrate of proliferating breast tumor cells as compared to control clonesin the presence or absence of tetracycline.

[0030]FIG. 3 is a graph showing the effects of overexpression of 95 onMCF7 induced serum deprivation (0.1%) cell death. T is tetracyclie and sis serum.

[0031]FIG. 4 is a graph showing the effects of 95 overexpression onprotection of MCF7 cells against ischemia-induced cell death. T istetracycline and I is ischemia.

[0032]FIG. 5 is a graph showing the effects of 95 overexpression onprotection of MCF7 cells against H₂O₂ (1 mM)-induced cell death. T istetracycline and H is H₂O₂.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0033] I. Definitions

[0034] The following definitions apply to the terms used in the presentspecification and claims:

[0035] The term “gene” refers to the genomic nucleotide sequence whichis transcribed to a full-length RNA. Such RNA molecules may be convertedinto corresponding cDNA molecules by techniques well known to the art ofrecombinant DNA technology. The term “gene” classically refers to thegenomic sequence, which, upon processing, can produce different RNAS,e.g., by splicing events. However, for ease of reading, any full-lengthcounterpart RNA sequence will also be referred to by shorthand herein asa “gene”.

[0036] The term “Expressed Sequence Tag” or “EST” refers to a partialDNA or cDNA sequence of about 150 to 500, more preferably about 300,sequential nucleotides of a longer sequence obtained from a genomic orcDNA library prepared from a selected cell, cell type, tissue type,organ or organism which longer sequence corresponds to an mRNA (or otherfull-length RNA) transcribed by a gene found in that library. In thiscase, the gene is found in rat neuronal cells. One or more librariesmade from a single tissue type typically provide at least about 3,000different (i.e., unique) ESTs and potentially the full complement of allpossible ESTs representing all cDNAs, e.g., 50,000-100,000 in an animalsuch as a human. Further background and information on the constructionof ESTs is described in Adams et al (1991) and International ApplicationNumber PCT/US92/05222 (Jan. 7, 1993).

[0037] The term “apoptosis” is particularly defined as the singledeletion of scattered cells by fragmentation into membrane-boundparticles which are phagocytosed by other cells, believed to be due toprogrammed cell death. However, as used herein, it should be understoodthat this term should be construed more broadly as encompassing neuronalcell death, whether or not that cell death is strictly by means theapoptotic process described above.

[0038] Two proteins are “cognate”, if they are produced in differentspecies, but are sufficiently similar in structure and biologicalactivity to be considered the equivalent proteins for those species. Twoproteins may also be considered cognate if they have at least 50% aminoacid sequence identity (when globally aligned with a pam250 scoringmatrix with a gap penalty of the form q+r (k−1) where k is the length ofthe gap, q=−12 and r=−4; percent identity=number of identities aspercentage of length of shorter sequence) and at least one biologicalactivity in common. Similarly, two genes are cognate if they areexpressed in different species and encode cognate proteins.

[0039] II. Novel Polynucleotide Sequences

[0040] The present invention identifies polynucleotides (nucleic acidsequences) with sequences as set forth herein in SEQ ID NOs: 1-12, thathave been significantly up-regulated when subjected to hypoxia. SEQ IDNOs: 1-4 and 8-12 have not previously been identified. SEQ ID NO 5 wasfound to match sequences in data banks but has not been reported to beassociated with hypoxia regulation.

[0041] To the extent that the positively identified sequence is a novelsequence, the present invention comprehends that novel sequence, as wellas any naturally-occurring polynucleotide that includes that sequence asa part thereof. The sequence per se has utility based on the fact thatit has been identified on the basis of differential expression in cellssubjected to hypoxic stress. It can be used in diagnostic processes andkits for determining whether any given cells have been subjected tohypoxic stress. Even when such sequences are rat sequences, i.e., SEQ IDNOs: 5 and 7, there is real-world utility for the purpose of medicalresearch for determining in a rat model which cells have been subjectedto hypoxic stress and which cells may have been protected from hypoxicstress when subjected to a treatment protocol in a rat model. By usingthe novel sequence as a probe, or a portion thereof as anoligonucleotide probe, one can identify the places in the organism(whether the organism is a rat when the sequence is a rat sequence or ahuman when the sequence is a human sequence) where the cDNA includingthe sequence is expressed and whether or not, or in what degree, it isexpressed when subjected to various treatment protocols.

[0042] Human genes may be discovered by determining the human gene whichcorresponds to the rat gene discovered in accordance with the presentinvention. Such human genes are also useful for determining whetherhuman cells have been subjected to hypoxic stress, for example indiagnosing whether or not a patient has suffered a stroke. As will bediscussed in greater detail below, it is a procedurally routine matterto determine a cognate human gene based on the sequence of a rat gene.Thus, regardless of whether or not one knows the actual sequence of thecorresponding human gene, the rat gene has utility as a probe forseeking and identifying the corresponding human gene which, whenidentified, will have its own utility.

[0043] The positively identified polynucleotide sequences are ESTs. Thelocation of an EST in a full-length cDNA is determined by analyzing theEST for the presence of coding sequence. A conventional computer programis used to predict the extent and orientation of the coding region of asequence (using all six reading frames). Based on this information, itis possible to infer the presence of start or stop codons within asequence and whether the sequence is completely coding or completelynon-coding or a combination of the two. If start or stop codons arepresent, then the EST can cover both part of the 5′-untranslated or3′-untranslated part of the mRNA (respectively) as well as part of thecoding sequence. If no coding sequence is present, it is likely that theEST is derived from the 3′ untranslated sequence due to its longerlength and the fact that most cDNA library construction methods arebiased toward the 3′ end of the mRNA. It should be understood that bothcoding and non-coding regions may provide ESTs equally useful in thedescribed invention.

[0044] As will be discussed below, even ESTs are directly useful as theyhave a length that allows for PCR (polymerase chain reaction), for useas a hybridization probe and have a unique designation for the gene withwhich it hybridizes (generally under conditions sufficiently stringentto require at least 95% base pairing). For a detailed description andreview of ESTs and their functional utility see, WO 93/00353 PCTApplication which is incorporated herein in its entirety by reference,as well as the references by Zweiger et al, 1997; Okubo et al, 1997 andBraren et al, 1997.

[0045] The WO 93/00353 PCT application further describes how the ESTsequences can be used to identify the transcribed genes.

[0046] Methods for obtaining complete gene sequences from ESTs arewell-known to those of skill in the art. See, generally, Sambrook et al,(1989) and Ausubel et al (1994-2000). Briefly, one suitable methodinvolves purifying the DNA from the clone that was sequenced to give theEST and labeling the isolated insert DNA. Suitable labeling systems arewell known to those of skill in the art. See, e.g., Davis et al (1986).The labeled EST insert is then used as a probe to screen a lambda phagecDNA library or a plasmid cDNA library, identifying colonies containingclones related to the probe cDNA that can be purified by known methods.The ends of the newly purified clones are then sequenced to identifyfull-length sequences and complete sequencing of full-length clones isperformed by enzymatic digestion or primer walking. A similar screeningand clone selection approach can be applied to clones from a genomic DNAlibrary. The entire naturally-occurring cDNA or gene sequence, includingany allelic variations thereof, all will have the same utility asdiscussed above for the identified polynucleotide.

[0047] The complete gene sequence of naturally-occurring variants of thegene in question, such as, for example, allelic variations, may bedetermined by hybridization of a cDNA library using a probe which isbased on the identified polynucleotide, under highly stringentconditions or under moderately stringent conditions. Stringencyconditions are a function of the temperature used in the hybridizationexperiment and washes, the molarity of the monovalent cations in thehybridization solution and in the wash solution(s) and the percentage offormamide in the hybridization solution. In general, sensitivity byhybridization with a probe is affected by the amount and specificactivity of the probe, the amount of the target nucleic acid, thedetectability of the label, the rate of hybridization, and the durationof the hybridization. The hybridization rate is maximized at a Ti(incubation temperature) of 20-25° C. below Tm for DNA:DNA hybrids and10-15° C. below Tm for DNA:RNA hybrids. It is also maximized by an ionicstrength of about 1.5M Na⁺. The rate is directly proportional to duplexlength and inversely proportional to the degree of mismatching.

[0048] Specificity in hybridization, however, is a function of thedifference in stability between the desired hybrid and “background”hybrids. Hybrid stability is a function of duplex length, basecomposition, ionic strength, mismatching, and destabilizing agents (ifany).

[0049] The Tm of a perfect hybrid may be estimated for DNA:DNA hybridsusing the equation of Meinkoth et al (1984), as

Tm=81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L

[0050] and for DNA:RNA hybrids, as

Tm=79.8° C.+18.5 (log M)+0.58 (%GC)−11.8 (%GC) ²−0.56(% form)−820/L

[0051] where

[0052] M, molarity of monovalent cations, 0.01-0.4 M NaCl,

[0053] %GC, percentage of G and C nucleotides in DNA, 30%-75%,

[0054] % form, percentage formamide in hybridization solution, and

[0055] L, length hybrid in base pairs.

[0056] Tm is reduced by 0.5-1.5° C. (an average of 1° C. can be used forease of calculation) for each 1% mismatching.

[0057] The Tm may also be determined experimentally. As increasinglength of the hybrid (L) in the above equations increases the Tm andenhances stability, the full-length rat gene sequence can be used as theprobe.

[0058] Filter hybridization is typically carried out at 68° C., and athigh ionic strength (e.g., 5-6×SSC), which is non-stringent, andfollowed by one or more washes of increasing stringency, the last onebeing of the ultimately desired stringency. The equations for Tm can beused to estimate the appropriate Ti for the final wash, or the Tm of theperfect duplex can be determined experimentally and Ti then adjustedaccordingly.

[0059] Hybridization conditions should be chosen so as to permit allelicvariations, but avoid hybridizing to other genes. In general, stringentconditions are considered to be a Ti of 5° C. below the Tm of a perfectduplex, and a 1% divergence corresponds to a 0.5-1.5° C. reduction inTm. Typically, rat clones were 95-100% identical to database ratsequences, and the observed sequence divergence may be artifactual(sequencing error) or real (allelic variation). Hence, use of a Ti of5-15° C. below, more preferably 5-10° C. below, the Tm of the doublestranded form of the probe is recommended for probing a rat cDNA librarywith rat EST probes. However, when probing for a human gene cognate,more moderate stringency hybridization conditions should be used.

[0060] As used herein, highly stringent conditions are those which aretolerant of up to about 15% sequence divergence, while moderatelystringent conditions are those which are tolerant of up to about 30-35%sequence divergence. Without limitation, examples of highly stringent(5-15° C. below the calculated Tm of the hybrid) and moderatelystringent (15-20° C. below the calculated Tm of the hybrid) conditionsuse a wash solution of 0.1×SSC (standard saline citrate) and 0.5% SDS atthe appropriate Ti below the calculated Tm of the hybrid. The ultimatestringency of the conditions is primarily due to the washing conditions,particularly if the hybridization conditions used are those which allowless stable hybrids to form along with stable hybrids. The washconditions at higher stringency then remove the less stable hybrids. Acommon hybridization condition that can be used with the highlystringent to moderately stringent wash conditions described above ishybridization in a solution of 6×SSC (or 6×SSPE), 5× Denhardt's reagent,0.5% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA at anappropriate incubation temperature Ti.

[0061] Once any such naturally-occurring DNA is identified, it can betested by means of routine experimentation to determine whether it isdifferentially expressed in the cells in which it naturally occurs whensubjected to hypoxic stress. The present invention is intended tocomprehend any such naturally-occurring DNA which binds to an EST of thepresent invention or any oligonucleotide fragment thereof, preferablyhaving at least 20, more preferably at least 50, contiguous nucleicacids, under highly stringent conditions or under moderately stringentconditions, which identified DNA molecules are determined to bedifferentially expressed in the cells in which they naturally occur whensuch cells are subjected to hypoxic stress. Any such identified DNAmolecules would have the same utility as discussed above for theidentified polynucleotide.

[0062] If the full-length sequence identified is a rat gene sequence ora sequence of any mammalian gene other than human, the cognate humangene sequence can be readily obtained, as would be readily appreciatedby those of skill in the art. Comparison of known cognate protein andgene sequences between rat and human shows a high level of sequenceidentity, mostly on the order of 70% or higher. The cognate human genesequence is quite readily identified and determined as long as there isa high level of sequence identity to the rat gene sequence.

[0063] While a rat EST sequence would be used to probe a rat cDNAlibrary for a full-length cDNA sequence, and could even be used to probehuman cDNA libraries, it would be expected that there would be somesequence divergence, especially at the EST sequence level, betweencognate rat and human DNAs, which sequence divergence may be possibly asmuch as 25-50w. Preferably, the rat sequence used as a probe is from thecoding region of the rat cDNA, as 5′- or 3′-uncoded region often lacksignificant homology among different mammalian species.

[0064] If a partial human cDNA is obtained, it may be used to isolate alarger human cDNA, and the process repeated as needed until the completehuman cDNA is obtained.

[0065] For cross-species hybridization, such as to obtain the cognatehuman gene sequence from the rat gene sequence, the Ti should be reducedfurther, by about 0.5-1.5° C., e.g., 1° C., for each expected lodivergence in sequence. The degree of divergence may be estimated fromthe known divergence of the most closely related pairs of known genesfrom the two species.

[0066] If the desired degree of mismatching results in a washtemperature less than 45° C., it is desirable to increase the saltconcentration so a higher temperature can be used. Doubling the SSCconcentration results in about a 17° C. increase in Tm, so washes at 45°C. in 0.1×SSC and 62° C. in 0.2×SSC are equivalent (1×SSC=0.15 M NaCl,0.015M trisodium citrate, pH 7.0).

[0067] The person skilled in the art can readily determine suitablecombinations of temperature and salt concentration to achieve thesedegrees of stringency.

[0068] Examples of successful cross-species-hybridization experimentsinclude Braun et al (1989) (mouse v. human), Imamura et al (1991) (humanv. rat), Oro et al (1988) (human v. Drosophila), Higuti et al (1991)(rat v. human), Jeung et al (1992) (rat, bovine v. human), Iwata et al(1992) (human v. mouse), Libert et al (1992) (dog v. human), Wang et al(1993) (human v. mouse), Jakubiczka et al (1993) (human v. bovine),Nahmias et al (1991) (human v. mouse), Potier et al (1992) (rat v.human), Chan et al (1989) (human v. mouse), Hsieh et al (1989) (human,mouse v. bovine), Sumimoto et al (1989) (human v. mouse), Boutin et al(1989) (rat v. human), He et al (1990) (human, rat v. dog, guinea pig,frog, mouse), Galizzi et al (1990) (mouse v. human). See also Gould etal (1989).

[0069] In general, for cross-species hybridization, Ti=25-35° C. belowTm. Wash temperatures and ionic strengths may be adjusted empiricallyuntil background is low enough.

[0070] Any non-rat mammalian sequences obtained from such hybridizationexperiments, which sequences test positive for the ability to bedifferentially expressed when the cells in which they naturally occurare subjected to hypoxic stress, are also encompassed by the presentinvention as are any non-human mammalian sequences obtained from suchhybridization experiments using the human gene as a probe to findcognate non-human mammalian genes.

[0071] Fragments of any such naturally-occurring sequences also haveutility and are intended to be encompassed by the present invention.Fragments of preferably at least 20, more preferably at least 50,nucleotides in length can be used as probes for the diagnostic assaysdescribed above.

[0072] Polynucleotide sequences that are complementary to any of thesequences or fragments encompassed by the present invention discussedabove are also considered to be part of the present invention. Wheneverany of the sequences discussed above are produced in a cell, thecomplementary sequence is concomitantly produced and, thus, thecomplementary sequence can also be used as a probe for the samediagnostic purposes.

[0073] Modifications or analogs of polynucleotides can be introduced toimprove the therapeutic properties of the polynucleotides. Improvedproperties include increased nuclease resistance and/or increasedability to permeate cell membranes.

[0074] Nuclease resistance, where needed, is provided by any methodknown in the art that does not interfere with biological activity of theantisense oligodeoxy-nucleotides, cDNA and/or ribozymes as needed forthe method of use and delivery (Iyer et al, 1990; Eckstein, 1985;Spitzer et al, 1988; Woolf et al, 1990; Shaw et al, 1991). Modificationsthat can be made to oligonucleotides in order to enhance nucleaseresistance include modifying the phosphorous or oxygen heteroatom in thephosphate backbone. These include preparing methyl phosphonates,phosphorothioates, phosphorodithioates and morpholino oligomers. In oneembodiment it is provided by having phosphorothioate bonds linkingbetween the four to six 3′-terminus nucleotide bases. Alternatively,phosphorothioate bonds link all the nucleotide bases. Othermodifications known in the art can be used where the biological activityis retained, but the stability to nucleases is substantially increased.

[0075] The present invention also includes all analogs of, ormodifications to, a polynucleotide of the invention that does notsubstantially affect the function of the polynucleotide. The nucleotidescan be selected from naturally occurring or synthetic modified bases.Naturally occurring bases include adenine, guanine, cytosine, thymineand uracil. Modified bases of the oligonucleotides include xanthine,hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-azathymine, pseudo uracil, 4-thiuracil, 8-halo adenine, 8-aminoadenine,8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiolguanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other substitutedguanines, other aza and deaza adenines, other aza and deaza guanines,5-trifluoromethyl uracil and 5-trifluoro cytosine.

[0076] In addition, analogs of nucleotides can be prepared wherein thestructure of the nucleotide is fundamentally altered and that are bettersuited as therapeutic or experimental reagents. An example of anucleotide analog is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA0 is replacedwith a polyamide backbone which is similar to that found in peptides.PNA analogs have been shown to be resistant to degradation by enzymesand to have extended lives in vivo and in vitro. Further, PNAs have beenshown to bind stronger to a complementary DNA sequence than a DNAmolecule. This observation is attributed to the lack of charge repulsionbetween the PNA strand and the DNA strand. Other modifications that canbe made to oligonucleotides include polymer backbones, cyclic backbones,or acyclic backbones.

[0077] III. Novel Proteins Encoded by Genes of Section II

[0078] Once the sequence of any full-length cDNA is obtained, theprotein encompassed thereby is readily determinable by analysis of thesequence to find the start and stop codons and then decoding the aminoacid sequence encoded by the cDNA. Thus, the present invention alsoencompasses any protein encoded by a full-length cDNA encompassed by thepresent invention as discussed above. Such proteins can be used for thesame diagnostic utility, as discussed above for the polynucleotides, asthey will be differentially expressed to the same degree that thecorresponding cDNA is differentially expressed. They can be used to makea diagnostic tool which can be used to determine their presence in acell. Thus, for example, they can be used to raise antibodies that couldbe used in such a diagnostic assay for the presence of such a protein.Such an assay would be useful to determine whether any given cell hadbeen subjected to neurotoxic stress. Such proteins can also be used forany of the utilities discussed hereinbelow in the section related tomethods of use.

[0079] Analogs of a protein or polypeptide encoded by the DNA sequencesdiscovered in the assays described herein is also comprehended by thepresent invention. Preferably, the analog is a variant of the nativesequence which has an amino acid sequence having at least 70% identityto the native amino acid sequence and retains the biological activitythereof. More preferably, such a sequence has at least 85% identity, atleast 90% identity, or most preferably at least 95% identity to thenative sequence.

[0080] The term “sequence identity” as used herein means that thesequences are compared as follows. The sequences are aligned usingversion 9 of the Genetic Computing Group's GAP (global alignmentprogram), using the default (BLOSUM62) matrix (values −4 to +11) with agap open penalty of −12 (for the first null of a gap) and a gapextension penalty of −4 (per each additional consecutive null in thegap). After alignment, percentage identity is calculated by expressingthe number of matches as a percentage of the number of amino acids inthe claimed sequence.

[0081] Analogs in accordance with the present invention may also bedetermined in accordance with the following procedure. Polypeptidesencoded by any nucleic acid, such as DNA or RNA, which hybridize to thecomplement of the native DNA or RNA under highly stringent or moderatelystringent conditions, as long as that polypeptide maintains thebiological activity of the native sequence are also considered to bewithin the scope of the present invention. Preferably, such nucleicacids hybridizing to the complement of the polynucleotides of thepresent invention under the specified conditions are naturally occurringnucleic acids, which may or may not be produced in cells of the samespecies as the original polynucleotides. As with any other analog, suchpolypeptide must retain the biological activity of the originalpolypeptide.

[0082] The term “active fragments” is intended to cover any fragment ofthe proteins identified by means of the present invention that retainthe biological activity of the full protein. For example, fragments canbe readily generated from the full protein where successive residues canbe removed from either or both the N-terminus or C-terminus of theprotein, or from biologically active peptides obtained therefrom byenzymatic or chemical cleavage of the polypeptide. Thus, multiplesubstitutions are not involved in screening for active fragments. If theremoval of one or more amino acids from one end or the other does notaffect the biological activity after testing in the standard tests,discussed herein, such truncated polypeptides are considered to bewithin the scope of the present invention. Further truncations can thenbe carried out until it is found where the removal of another residuedestroys the biological activity.

[0083] “Functional derivatives” as used herein covers chemicalderivatives which may be prepared from the functional groups which occuras side chains on the residues or the N- or C-terminal groups, by meansknown in the art, and are included in the invention as long as theyremain pharmaceutically acceptable, i.e., they do not destroy thebiological activity of the corresponding protein as described herein anddo not confer toxic properties on compositions containing it.Derivatives may have chemical moieties, such as carbohydrate orphosphate residues, provided such a fraction has the same biologicalactivity and remains pharmaceutically acceptable.

[0084] Suitable derivatives may include aliphatic esters of the carboxylof the carboxyl groups, amides of the carboxyl groups by reaction withammonia or with primary or secondary amines, N-acyl derivatives or freeamino groups of the amino acid residues formed with acyl moieties (e.g.,alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of freehydroxyl group (e.g., that of seryl or threonyl residues) formed withacyl moieties. Such derivatives may also include for example,polyethylene glycol side-chains which may mask antigenic sites andextend the residence of the complex or the portions thereof in bodyfluids.

[0085] Non-limiting examples of such derivatives are described below.

[0086] Cysteinyl residues most commonly are reacted withalpha-haloacetates (and corresponding amines), such as chloroacetic acidor chloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, alpha-bromo- beta-(5-imidazoyl)propionic acid,chloroacetyl phosphate, B alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl-2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri−4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0087] Histidyl residues are derivatized by reaction withdiethylprocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Parabromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0.

[0088] Lysinyl and amino terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing alpha-amino-containing residuesinclude imidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2, 4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

[0089] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3- butanedione,1,2-cyclodexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

[0090] The specific modification of tyrosyl residues per se has beenstudied extensively, with particular interest in introducing spectrallabels into tyrosyl residues by reaction with aromatic diazoniumcompounds or tetranitromethane. Most commonly, N-acetylimidazole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

[0091] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-[2-morpholinyl-(4-ethyl)]carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethlypentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

[0092] Glutaminyl and asparaginyl residues are frequently deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

[0093] The term “derivatives” is intended to include only thosederivatives that do not change one amino acid to another of the twentycommonly-occurring natural amino acids.

[0094] The term “salts” herein refers to both salts of carboxyl groupsand to acid addition salts of amino groups of the complex of theinvention or analogs thereof. Salts of a carboxyl group may be formed bymeans known in the art and include inorganic salts, for example, sodium,calcium, ammonium, ferric or zinc salts, and the like, and salts withorganic bases as those formed, for example, with amines, such astriethanolamine, arginine or lysine, piperidine, procaine and the like.Acid addition salts include, for example, salts with mineral acids, suchas, for example, hydrochloric acid or sulfuric acid, and salts withorganic acids, such as, for example, acetic acid or oxalic acid. Ofcourse, any such salts must have substantially similar biologicalactivity to the complex of the invention or its analogs.

[0095] IV. Known Polynucleotides and Protein Sequences

[0096] To the extent that any of the polynucleotide sequences of thepresent invention are determined to appear in the sequence databanks andmay be part of identified known genes with known function and encodeknown proteins, it is not necessary to go through the hybridizationsteps in order to find the full-length CDNA for such ESTs. Furthermore,in most cases, it will not be necessary to find the cognate human geneexperimentally. If the rat EST is part of a known rat gene, it is likelythat the cognate human gene is also known. If not, it may be determinedby the techniques discussed hereinabove with respect to novel rat genesequences.

[0097] As the protein encoded by the known gene is also known, it is notnecessary to use the techniques discussed hereinabove for determiningthe sequence encoded by a polynucleotide sequence. However, to theextent that the protein is not known, the techniques discussedhereinabove with respect to novel polynucleotide sequences may also beused.

[0098] Any known allelic variants of the known gene would also beexpected to have the properties discovered by the gene discoverytechniques discussed herein and, therefore, are also considered to bepart of the present invention. The existence of othernaturally-occurring variants having the property of having its sequencemodulated when subjected to neurotoxic stress may also be determinedusing hybridization experiments under highly stringent conditions ormoderately stringent conditions, all as discussed in detail hereinabovewith respect to the novel polynucleotide sequences.

[0099] Analogs, active fragments, functional derivatives and salts ofthe known proteins which retain the property of that protein for thepurposes of the present invention (although not necessarily for theproperties previously known for that protein) are comprehended by thepresent invention, if novel, and their use is considered to be part ofthe present invention.

[0100] V. Utility of Good Genes and Bad Genes

[0101] The genes found to be differentially expressed when the cellproducing them are subjected to hypoxic stress, may be genes whichcontribute to the adverse effects of hypoxia, such as apoptosis, and, insome circumstances, angiogenesis, or genes which contribute to thealleviation of the detrimental effects of hypoxia. The former genes,which contribute to the adverse effects of hypoxia will be referred toas “bad genes” herein. It would be desirable to down-regulate orotherwise decrease the titre of the expression product of such bad genesat the site of the hypoxic event, such as stroke. The utility of suchbad genes and methods of use thereof will be discussed below.

[0102] Those genes which contribute to the alleviation of thedetrimental effects of hypoxia, including avoidance of apoptosis andcausing angiogenesis, will be referred to herein as “good genes”. Itwould be desirable to up-regulate or otherwise increase the titres ofthe expression product of such good genes at the site of the hypoxiaevent. The utility of such good genes and methods of use thereof will bediscussed below.

[0103] While it is not possible to directly determine from thedifferential expression studies in which tehse genes were found whetherthe DNA fragmenets found are part of a good or bad gene, it isreasonably certain that the fragments so identified are one or the otheras their expression has been significantly modulated based on thehypoxia stress conditions to which the cells have been subjected.However, by means of further experimentation, which experimentationwould not be considered to be undue experimentation, one can determinewhether the fragments are part of good genes or bad genes. One way totest it is to create a mutation on the ATG codon of the fragment orcreate a frame shift mutation and then check whether its effect is thesame. If the effect is different, it is a peptide which causes theeffect. If the effect is the same, it is not a peptide but the RNAitself which causes the activity. Another possibility is to create asynthetic peptide and introduce it into cells to check whether it showsthe relevant phenotype.

[0104] Another way to test whether the fragments are part of good genesor bad genes is to knock out the gene of interest, either in an animalwith a knockout gene or by knocking out the gene in the cell line beingtested. In a cell line, the cells can then be tested with hypoxic stressto determine whether the absence of that gene has a protective effect orenhances cell death. In a knockout mouse, similar tests can be conductedto see whether the absence of that gene has a protective or detrimentaleffect on the mouse when subjected to hypoxic stress.

[0105] A gene can be knocked out in a cell line by means of homologousrecombination or by transfecting the cell line with an antisensesequence which prevents the expression of that gene, all as is wellknown to those of ordinary skill in this art. A gene can be knocked outin an animal such as a mouse, by the techniques discussed below.

[0106] Accordingly, even if it cannot be directly determined whether anyof the specific DNA fragments of the present invention are parts of goodgenes or parts of bad genes, it is reasonably expected that they areparts of either one or the other, and, in either event, they haveutility for the reasons discussed below. It can be determined whetherthey are good genes or bad genes without resorting to undueexperimentation. Accordingly, such genes have utility and industrialapplicability.

[0107] Good genes are useful as the protein encoded by such genes can beused to protect neural from, and ameliorate the effects of, hypoxia andischemia, and ultimately in the therapeutic treatment of stroke, hypoxiaand/or ischemia. Such genes may prevent apoptosis or promoteangiogenesis. As to the latter, promotion of angiogenesis may bedesirable, for example, in trauma situations where a limb must bereattached or in a transplant where revascularization is needed. Thusthe genes, and the DNA encoding such a protein or active fragment oranalog thereof, are useful in the recombinant production of suchproteins or polypeptides. They are also useful as a target for assaysfor the discovery of drugs which selectively up-regulate such genes. Theproteins encoded by such novel good genes, as well as active fragmentsthereof, analogs and functional derivatives thereof, are also part ofthe present invention and have utility to protect cells from, and toameliorate the effects of, hypoxia and ischemia, and ultimately in thetherapeutic treatment of stroke, hypoxia ischemia, and/or otherconditions where such effects would be desirable.

[0108] It may turn out that the beneficial effect of up-regulation of agood gene is due to the production of a non-protein product of thegene's activity. Even in that case, however, up-regulation of the goodgene will cause enhanced production of that product.

[0109] Good genes, whether novel or known, but whose relationship tohypoxia reported herein was previously unknown, may be used in novelprocesses which take advantage of these newly discovered properties.Thus, for example, the expression product of such genes, as well asactive fragments, analogs and functional derivatives thereof, may beused to protect cells from the adverse effects of hypoxia or ischemia,to ameliorate the effects of hypoxia or ischemia, and ultimately for thetreatment of the effects of stroke, hypoxia, ischemia, and/or otherconditions where such effects would be desirable, by the therapeuticadministration thereof in a manner which causes such product to bebrought into the vicinity of the cells to be treated.

[0110] Bad genes are useful in that they can be used in diagnosticassays for cells that have been subjected to hypoxia or ischemia. IfmRNA corresponding to such genes, or the translation product thereof, isfound in the cells being assayed it is likely that they have beensubjected to hypoxia or ischemia. If diagnosed pre-stroke, this may bepredictive of incipient stroke. They are also useful as a target forassays for the discovery of drugs which selectively down-regulate suchgenes or are otherwise dominant negative with respect to the expressionof the gene product of such genes. Antisense RNA that prevents theexpression of such gene is also part of the present invention and isuseful to protect neural cells from neurotoxicity, to ameliorate theeffects of hypoxia or ischemia, and ultimately for the treatment of theeffects of stroke, hypoxia and/or ischemia. The bad gene may also beused therapeutically when these “bad” effects may be useful for treatinga certain condition. For example, promotion of apoptosis may be usefulfor removing unwanted cells, such as tumor cells. Prevention ofangiogenesis may also be useful under certain circumstances.

[0111] It may turn out that the detrimental effect of up-regulation of abad gene is due to the production of non-protein product of the gene'sactivity. Even in that case, however, down-regulation of the bad genewill cause diminished production of that product.

[0112] Bad genes, whether novel or known but whose relationship tohypoxia reported herein was previously unknown, may be used in novelprocesses which take advantage of these newly discovered properties.Antisense RNA having a sequence complementary to a portion of such geneand that prevents the expression of such gene may be produced and usedtherapeutically by administering same in a manner by which it enterscells which have been subjected to stroke, hypoxia, and/or ischemia inorder to ameliorate the effects of such conditions. They may also beused in methods for assaying for drugs which down-regulate such genes.To the extent that such proteins are enzymes, the present inventioncomprehends the protection of neural cells from neurotoxicity, theamelioration of the effects of hypoxia or ischemia, and ultimately thetherapeutic treatment of the effects of stroke, hypoxia and/or ischemiaby administering an inhibitor of such enzyme in a manner that bringssuch inhibitor to the vicinity of the cells in which such enzyme hasbeen up-regulated.

[0113] VI. Diagnostic Methods

[0114] As all of the genes of the present invention have been found tobe modulated significantly upward after the cells have been subject tohypoxia, all of such genes may be considered to be a gene of interestfor the purpose of the diagnostic assays reported herein.

[0115] Methods of detecting tissue hypoxia in mammalian tissue are basedon the use of the mRNA of the genes of interest or the translationproduct thereof as a diagnostic marker for cells that have beensubjected to hypoxia or ischemia. It is possible to determine the levelof the mRNAs or protein translation products corresponding to these badgenes, in normal tissue or bodily fluids as compared to hypoxic tissue abodily fluid from a subject which has suffered a hypoxic event, and,thus, determine the reference values of these genes on mRNAs or proteinswhich are indicative of tissue hypoxia. For identification of the gene,in situ hybridization, Southern blotting, single strand conformationalpolymorphism, restriction endonuclease fingerprinting (REF), PCRamplification and DNA-chip analysis using the nucleic acid sequences ofthe present invention as probes/primers can be used.

[0116] Methods of obtaining tissue samples for analysis include anysurgical and non-surgical technique known in the art. Surgical methodsinclude, but are not limited to biopsy such as fine needle aspirate,core biopsy, dilation and curettage.

[0117] Samples

[0118] The sample for use in the detection methods may be of anybiological fluid or tissue which is reasonably expected to contain themessenger RNA transcribed from one of the above genes of interest, or aprotein expressed therefrom one of the above bad genes. The bodilyfluids can include tears, serum, urine, sweat or other bodily fluidwhere secreted proteins from the tissue that is undergoing an ischemicevent can be localized. Preferably, the sample is composed of cells fromthe subject being tested which are suspect of having been subjected to ahypoxic event, such as neural cells from a suspected stroke area orcardiac cells from a suspect infarct area.

[0119] Analyte Binding Reagents

[0120] The assay target or analyte as a diagnostic marker may be anucleic acid, such as mRNA of a gene of interest, or a proteintranslation product thereof. When the assay target is a nucleic acid,the preferred binding reagent is a complementary nucleic acid. However,the nucleic acid binding agent may also be a peptide or protein. Apeptide phage library may be screened for peptides which bind thenucleic acid assay target. In a similar manner, a DNA binding proteinmay be randomly mutagenized in the region of its DNA recognition site,and the mutants screened for the ability to specifically bind thetarget. Or the hypervariable regions of antibodies may be mutagenizedand the antibody mutants displayed on phage.

[0121] When the assay target is a protein, the preferred binding reagentis an antibody, the specifically binding fragment of an antibody, or amolecule that has the antigen-binding portion of an antibody. Theantibody may be monoclonal or polyclonal. It can be obtained by firstimmunizing a mammal with the protein target, and recovering eitherpolyclonal antiserum, or immunocytes for later fusion to obtainhybridomas, or by constructing an antibody phage library and screeningthe antibodies for binding to the target. The binding reagent may alsobe a binding molecule other than an antibody, such as a receptorfragment, an oligopeptide, or a nucleic acid. A suitable oligopeptide ornucleic acid may be identified by screening a suitable random library.

[0122] Signal Producing System (SPS)

[0123] In order to detect the presence, or measure the amount, of ananalyte, the assay must provide for a signal producing system (SPS) inwhich there is a detectable difference in the signal produced, dependingon whether the analyte is present or absent (or, in a quantitativeassay, on the amount of the analyte). The detectable signal may be onewhich is visually detectable, or one detectable only with instruments.Possible signals include production of colored or luminescent products,alteration of the characteristics (including amplitude or polarization)of absorption or emission of radiation by an assay component or product,and precipitation or agglutination of a component or product. The term“signal” is intended to include the discontinuance of an existingsignal, or a change in the rate of change of an observable parameter,rather than a change in its absolute value. The signal may be monitoredmanually or automatically.

[0124] Labels

[0125] The component of the signal producing system which is mostintimately associated with the diagnostic reagent for the analyte iscalled the “label”. A label may be, e.g., a radioisotope, a fluorophore,an enzyme, a co-enzyme, an enzyme substrate, an electron-dense compound,an agglutinable particle, etc.

[0126] The radioactive isotope can be detected by such means as the useof a gamma counter or a scintillation counter or by autoradiography.Isotopes which are particularly useful for the purpose of the presentinvention are ³H, ³²P, ¹²⁵I, ¹³¹ I, ³⁵S and ¹⁴C.

[0127] Diagnostic kits are also within the scope of this invention. Suchkits include monoclonal antibodies or nucleic acid probes that canrapidly detect tissue hypoxia.

[0128] For nucleic acid probes, the radioactive labeling can be carriedout according to any conventional method such as terminal labeling atthe 3′ or 5′ position with the use of a radiolabeled nucleotide, apolynucleotide kinase (with or without dephosphorylation by aphosphatase) or a ligase (according to the extremity to be labeled). Theprobes can be the matrix for the synthesis of a chain consisting ofseveral radioactive nucleotides or of several radioactive andnon-radioactive nucleotides. The probes can also be prepared by achemical synthesis using one or several radioactive nucleotides. Anothermethod for radioactive labeling is a chemical iodination of the probesof the invention which leads to the binding of several ¹²⁵I atoms on theprobes.

[0129] The label may also be a fluorophore. When the fluorescentlylabeled reagent is exposed to light of the proper wavelength, itspresence can then be detected due to fluorescence. Among the mostcommonly used fluorescent labeling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine.

[0130] Alternatively, fluorescence-emitting metals such as ¹²⁵Eu, orothers of the lanthanide series, may be incorporated into a diagnosticreagent using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) of ethylenediamine-tetraaceticacid (EDTA).

[0131] The label may also be a chemiluminescent compound. The presenceof the chemiluminescently labeled reagent is then determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of particularly useful chemiluminescentlabeling compounds are luminol, isolumino, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

[0132] Likewise, a bioluminescent compound may be used for labeling.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase and aequorin.

[0133] Enzyme labels, such as horseradish peroxidase and alkalinephosphatase, can also be used. When an enzyme label is used, the signalproducing system must also include a substrate for the enzyme. If theenzymatic reaction product is not itself detectable, the SPS willinclude one or more additional reactants so that a detectable productappears.

[0134] Conjugation Methods

[0135] A label may be conjugated, directly or indirectly (e.g., througha labeled anti-analyte binding reagent antibody), covalently (e.g., withN-succinimidyl 3-(2-pyridyldithio)propionate (SPDP)) or non-covalently,to the analyte binding reagent, to produce a diagnostic reagent.

[0136] Similarly, the analyte binding reagent may be conjugated to asolid phase support to form a solid phase (“capture”) diagnosticreagent.

[0137] Suitable supports include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention.

[0138] The support material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toits target. Thus the support configuration may be spherical, as in abead, or cylindrical, as in the inside surface of a test tube, or theexternal surface of a rod. Alternatively, the surface may be flat suchas a sheet, test strip, etc.

[0139] Binding Assay Formats

[0140] Binding assays may be divided into two basic types, heterogeneousand homogeneous. In heterogeneous assays, the interaction between theaffinity molecule and the analyte does not affect the label, hence, todetermine the amount or presence of analyte, bound label must beseparated from free label. In homogeneous assays, the interaction doesaffect the activity of the label, and therefore analyte levels can bededuced without the need for a separation step.

[0141] In one embodiment, the analyte binding reagent is insolubilizedby coupling it to a macromolecular support, and analyte in the sample isallowed to compete with a known quantity of a labeled or specificallylabelable analyte analogue. The “analyte analogue” is a molecule capableof competing with analyte for binding to the analyte binding reagent,and the term is intended to include analyte itself. It may be labeledalready, or it may be labeled subsequently by specifically binding thelabel to a moiety differentiating the analyte analogue from analyte. Thesolid and liquid phases are separated, and the labeled analyte analoguein one phase is quantified. The higher the level of analyte analogue inthe solid phase, i.e., sticking to the analyte binding reagent, thelower the level of analyte in the sample.

[0142] In a “sandwich assay”, both an insolubilized analyte bindingreagent, and a labeled analyte binding reagent are employed. The analyteis captured by the insolubilized analyte binding reagent and is taggedby the labeled analyte binding reagent, forming a ternary complex. Thereagents may be added to the sample in either order, or simultaneously.The analyte binding reagents may be the same or different. The amount oflabeled analyte binding reagent in the ternary complex is directlyproportional to the amount of analyte in the sample.

[0143] The two embodiments described above are both heterogeneousassays. However, homogeneous assays are conceivable. The key is that thelabel be affected by whether or not the complex is formed.

[0144] Detection of Genes of Interest

[0145] Detection of the mRNA of the genes of interest may be done byNorthern blot analysis on tissue biopsies. Tissue samples from patientsmay be obtained and the total RNA extracted using RNAStat 60. The totalRNA sample may then be resolved on denaturing gel by electrophoresis andthen transferred onto a nylon membrane. After transfer of RNA onto themembrane, the membrane may then be used in hybridization with a suitableprobe, which may be a synthetic probe directed against a gene alreadyknown to be a marker, or which may be a cDNA probe prepared directlyfrom subtractive hybridization, wherein the fragment encoding the geneof interest, that is up-regulated in tissue hypoxia, will be labeled,preferably either radioactively with ³²p or non-radioactively with DIG(Digoxigenin). A negative control, such as one composed of RNA samplefrom normal tissue of normal subjects, may be resolved side by side withthe patients' sample, to determine quantitatively whether there is asignificant increase in the level of gene expression. Elevation of themessenger RNA transcript from this gene would imply the presence ofhypoxia, ischemia or other neurotoxic stress.

[0146] In a hybridization assay, a nucleic acid reagent is used as aprobe. For probe use, only one reagent is needed, and it may hybridizeto all or just a part of the target nucleic acid. optionally, more thanone probe may be used to increase specificity.

[0147] In probe-based assays, hybridizations may be carried out onfilters or in solutions. Typical filters are nitrocellulose, nylon, andchemically-activated papers. The probe may be double stranded or singlestranded, however, the double stranded nucleic acid will be denaturedfor binding.

[0148] Techniques for detecting a protein translation product ofinterest include, but are not limited to, immunoblotting or Westernblotting, ELISA, sandwich assays, fluorescence, or biotin or enzymaticlabeling with or without secondary antibodies.

[0149] Western blot analysis can be done on the tissue biopsies ortissue aspirates. This would involve resolving the proteins on anelectrophoretic gel, such as an SDS PAGE gel, and transferring theresolved proteins onto a nitrocellulose or other suitable membrane. Theproteins are incubated with a target binding molecule, such as anantibody.

[0150] This binding reagent may be labeled or not. If it is unlabeled,then one would also employ a secondary, labeled molecule which binds tothe binding reagent. One approach involves avidinating one molecule andbiotinylating the other. Another is for the secondary molecule to be asecondary antibody which binds the original binding reagent.

[0151] To improve detection of the specific protein, immunoprecipitationcan be conducted. This typically will involve addition of a monoclonalantibody against the protein of interest to samples, then allowing theIg-protein complex to precipitate after the addition of an affinity bead(ie antihuman Ig Sepharose bead). The immunoprecipitates will undergoseveral washings prior to transfer onto a nitrocellulose membrane. TheWestern blot analysis can be performed using another antibody againstthe primary antibody used.

[0152] There are a number of different methods of delivering theradiolabeled analyte binding reagent to the end-user in an amountsufficient to permit subsequent dynamic and/or static imaging usingsuitable radiodetecting devices. It may be administered by any meansthat enables the active agent to reach the agent's site of action in thebody of a mammal. Because proteins and nucleic acids are subject tobeing digested when administered orally, parenteral administration,i.e., intravenous, subcutaneous, orintramuscular, would ordinarily beused to optimize absorption of an analyte binding reagent, such as anantibody, which is a protein.

[0153] The dosage is the smallest amount capable of providing adiagnostically effective image, and may be determined by meansconventional in the art, using known radioimaging agents as a guide.

[0154] Typically, the imaging is carried out on the whole body of thesubject, or on that portion of the body or organ relevant to thecondition or disease under study. The amount of radiolabeled analytebinding reagent accumulated at a given point in time in relevant targetorgans can then be quantified.

[0155] A particularly suitable radiodetecting device is a scintillationcamera, such as a gamma camera. A scintillation camera is a stationarydevice that can be used to image distribution of radiolabeled analytebinding reagent. The detection device in the camera senses theradioactive decay, the distribution of which can be recorded. Dataproduced by the imaging system can be digitized. The digitizedinformation can be analyzed over time discontinuously or continuously.The digitized data can be processed to produce images, called frames, ofthe pattern of uptake of the radiolabeled analyte binding reagent in thetarget tissue/organ at a discrete point in time. In most continuous(dynamic) studies, quantitative data is obtained by observing changes indistributions of radioactive decay in the target tissue/organ over time.In other words, a time-activity analysis of the data will illustrateuptake through clearance of the radiolabeled binding protein by thetarget organs with time.

[0156] Various factors should be taken into consideration in selectingan appropriate radioisotope. The radioisotope must be selected with aview to obtaining good quality resolution upon imaging, should be safefor diagnostic use in humans and animals (except for animal models whichwill be sacrificed thereafter and will be maintained anaesthetized untilthen), and should preferably have a short physical half-life so as todecrease the amount of radiation received by the body (with the sameexceptions). The radioisotope used should preferably bepharmacologically inert, and, in the quantities administered, should nothave any substantial physiological effect.

[0157] The analyte binding reagent may be radiolabeled with differentisotopes of iodine, for example ¹²³I, ¹²⁵I, or ¹³¹I (see for example,U.S. Pat. No. 4,609,725). The extent of radiolabeling must, however bemonitored, since it will affect the calculations made based on theimaging results (i.e., a diiodinated analyte binding reagent will resultin twice the radiation count of a similar monoiodinated analyte bindingreagent over the same time frame).

[0158] In applications to human subjects, it may be desirable to useradioisotopes other than ¹²⁵I for labeling in order to decrease thetotal dosimetry exposure of the human body and to optimize thedetectability of the labeled molecule (though this radioisotope can beused if circumstances require). Ready availability for clinical use isalso a factor. Accordingly, for human applications, preferredradiolabels are for example, ⁹⁹MTc, ⁶⁷Ga, ⁶⁸Ga, ⁹⁰Y, ¹¹¹In, ^(113m)In,¹²³I, ¹⁸⁶Re, ¹⁸⁸Re or ²¹¹At.

[0159] The radiolabeled analyte binding reagent may be prepared byvarious methods. These include radiohalogenation by the chloramine-Tmethod or the lactoperoxidase method and subsequent purification by HPLC(high pressure liquid chromatography), for example as described byGutkowska et al (1987). Other known method of radiolabeling can be used,such as IODOBEADS™.

[0160] For animal models, such as mice or rats, the animal may besacrificed after administration of the analyte binding reagent andregions which have been subjected to neurotoxic stress imaged onimmobilized brain slices.

[0161] VII. Screening Methods

[0162] Each of the genes identified by means of the present inventioncan be used as a candidate gene in a screening assay for identifying andisolating inhibitors of hypoxia or other neurotoxic stress. Many typesof screening assays are known to those of ordinary skill in the art. Thespecific assay which is chosen will depend to a great extent on theactivity of the candidate gene or the protein expressed thereby. Thus,if it is known that the expression product of a candidate gene hasenzymatic activity, then an assay which is based on inhibition of theenzymatic activity may be used. If the candidate protein is known tobind to a ligand or other interactor, then the assay can be based on theinhibition of such binding or interaction. When the candidate gene is aknown gene, then many of its properties will also be known, and thesecan be used to determine the best screening assay. If the candidate geneis novel, then some analysis and/or experimentation will be appropriatein order to determine the best assay to be used to find inhibitors ofthe activity of that candidate gene. The analysis may involve a sequenceanalysis to find domains in the sequence which would shed light on itsactivity. Other experimentation described herein to identify thecandidate gene and its activity, which experiment would not amount toundue experimentation, may also be engaged in so as to identify the typeof screen that would be appropriate to find inhibitors or enhancers, asthe case may be, for the candidate gene or the protein encoded thereby.

[0163] As is well known in the art, the screening assays may be in vivoor in vitro. An in vivo assay is a cell-based assay using any eukaryoticcell. One such cell-based system is particularly relevant in order todirectly measure the activity of candidate genes which are pro-apoptoticfunctional genes, i.e., expression of the gene will cause apoptosis orotherwise cause cell death in target cells. One way of running such anin vivo assay uses tetracycline-inducible (Tet-inducible) geneexpression. Tet-inducible gene expression is well known in the art(Hofmann et al, 1996). Tet-inducible retroviruses have been designedincorporating the Self-inactivating (SIN) feature of a 3′ Ltrenhancer/promoter retroviral deletion mutant. Expression of this vectorin cells is virtually undetectable in the presence of tetracycline orother active analogs. However, in the absence of Tet, expression isturned on to maximum within 48 hours after induction, with uniformincreased expression of the whole population of cells that harbor theinducible retrovirus, indicating that expression is regulated uniformlywithin the infected cell population.

[0164] When dealing with pro-apoptotic function candidate genes,Tet-inducible expression causes apoptosis in target cells. One canscreen for small molecules or peptides able to rescue the cells from thegene-triggered apoptosis.

[0165] If the gene product of the candidate gene phosphorylates with aspecific target protein, a specific reporter gene construct can bedesigned such that phosphorylation of this reporter gene product causesits activation, which can be followed by a color reaction. The candidategene can be specifically induced, using the Tet-inducible systemdiscussed above, and a comparison of induced vs. non-induced genesprovides a measure of reporter gene activation.

[0166] In a similar indirect assay, a reporter system can be designedthat responds to changes in protein-protein interaction of the candidateprotein. If the reporter responds to actual interaction with thecandidate protein, a color reaction will occur.

[0167] One can also measure inhibition or stimulation of reporter geneactivity by modulation of its expression levels via the specificcandidate promoter or other regulatory elements. A specific promoter orregulatory element controlling the activity of a candidate gene isdefined by methods well known in the art. A reporter gene is constructedwhich is controlled by the specific candidate gene promoter orregulatory elements. The DNA containing the specific promoter orregulatory agent is actually linked to the gene encoding the reporter.Reporter activity depends on specific activation of the promoter orregulatory element. Thus, inhibition or stimulation of the reporter willbe a direct assay of stimulation/inhibition of the reporter gene.

[0168] Various in vitro screening assays are also well within the skillof those of ordinary skill in the art. For example, if enzymaticactivity is to be measured, such as if the candidate protein has akinase activity, the target protein can be defined and specificphosphorylation of the target can be followed. The assay may involveeither inhibition of target phosphorylation or stimulation of targetphosphorylation, both types of assay being well known in the art.

[0169] One can also measure in vitro interaction of a candidate proteinwith interactors. In this screen, the candidate protein is immobilizedon beads. An interactor, such as a receptor ligand, is radioactivelylabeled and added. When it binds to the candidate protein on the bead,the amount of radioactivity carried on the beads (due to interactionwith the candidate protein) can be measured. The assay would indicateinhibition of the interaction by measuring the amount of radioactivityon the bead.

[0170] Any of the screening assays, according to the present invention,will include a step of identifying the small molecule or peptide whichtests positive in the assay and may also include the further step ofproducing that which has been so identified. The use of any suchmolecules identified for inhibiting hypoxia or other neurotoxic stressis also considered to be part of the present invention.

[0171] VIII. Therapeutic Methods Relating to Good Genes

[0172] In accordance with these findings, the present invention extendsto the treatment of stroke by the administration of astroke-ameliorating or stroke-inhibiting amount of an agent capable ofat least partially preventing brain damage, or averting the occurrenceor reducing the size and severity of an ischemic infarct due, forexample, to stroke, aneurysm, cerebrovascular accident, apoplexy orother trauma. Other conditions in which apoptosis is to be prevented orangiogenesis promoted may also be treatable by administration of thegood genes of the present invention. Exemplary situations where apromoter of angiogenesis would be useful include trauma situations wherea limb must be reattached or in a transplant where revascularization isneeded.

[0173] The present invention, therefore, extends to methods for thetreatment of stroke or other conditions caused or exacerbated by hypoxiaor ischemia as where apoptosis is to be prevented or angiogenesispromoted, and to corresponding pharmaceutical compositions, comprisingand including, without limitation, as active ingredients a proteinencoded by a good gene, as well as analogs, active fragments, functionalderivatives or salts thereof.

[0174] Within minutes after cessation of local cerebral blood flow, aregion of densely ischemic brain tissue becomes infarcted and dies. Thisinfarcted core is surrounded however, by a zone of ischemic butpotentially viable tissue termed the “ischemic penumbra,” which receivessuboptimal perfusion via collateral blood vessels. The volume of thepenumbra that ultimately becomes infarcted after an acute arterialocclusion is determined by a variety of factors that mediateneurotoxicity within this zone during the hours following interruptedblood flow. The nature of these factors (including glutamate, superoxideradicals, and nitric oxide) is only partially understood, as are thecomplex interactions that will determine whether ischemic tissue willdie or recover. Some of these factors are intrinsic to the locus ofischemia, and others are delivered to the penumbra via the circulation.The net result of signaling interactions between these factors cangreatly enhance neuronal cytotoxicity in the ischemic penumbra, causinga significantly larger volume of brain damage and necrosis, withcorresponding increases in functional damage. The good genes, inaccordance with the present invention, participate in mediatingincreased volumes of cerebral infarction during focal cerebral ischemia.

[0175] Good genes may also be used as the target of screening processesto find agents capable of enhancing the expression of a good gene. Thus,the amount of mRNA produced by a cell, before and after subjecting thecell to a neurotoxic stress, such as hypoxia, and administering a testagent, will determine whether that test agent causes further enhancementof expression of that good gene, as compared to a control in which notest agent is added. Such testing can reveal agents which are useful inthe treatment of stroke. Screening methods are discussed in Section VII,hereinabove.

[0176] IX. Therapeutic Methods Relating to Bad Genes

[0177] Bad genes may be used therapeutically for treating conditions inwhich promotion of apoptosis and/or inhibition of angiogenesis isdesirable. Promotion of apoptosis would be useful in treating tumorcells. Inhibition of angiogenesis may be useful, for example, withvascular stents where ingrowth is undesirable. The present invention,therefore, extends to methods for the treatment of cancer and otherconditions where promotion of apoptosis and/or inhibition ofangiogenesis is desired, and to corresponding pharmaceuticalcompositions, comprising and including, without limitation, as activeingredients a protein encoded by a bad gene, as well as analogs, activefragments, functional derivatives or salts thereof.

[0178] Additionally, the ability of an agent to inhibit expression ofbad genes provides an additional therapeutic mechanism in the treatmentof stroke since it would be expected to result in a reduction in thesize and severity of the infarction.

[0179] The present invention thus includes a method of screening for anagent capable of providing a neuroprotective effect and thus reducingthe size and severity of infarct size in stroke, which method comprisesadministering a test agent concurrent with, or subsequent to, aninfarct-producing amount of a product of a bad gene and measuring theresultant decrease in infarct size vis-a-vis a control dose of theinfarct-producing amount of the polyamine. Such testing can revealagents which are useful in the treatment of this aspect of stroke.Screening methods are discussed in Section VII, hereinabove.

[0180] The production and administration of antisense sequences andribozymes that specifically bind and cleave a particular mRNA sequenceare discussed in Sections XI and XII hereinafter. Such ribozymes andantisense sequences relating specifically to bad genes and the mRNA theydescribe will inhibit the expression of these bad genes and, thus, willprovide an additional therapeutic mechanism in treating the effects ofstroke, hypoxia and/or ischemia or other conditions in which apoptosisis to be inhibited and/or angiogenesis promoted. Similarly, negativedominant peptides are discussed in Section XIII. Such negative dominantpeptides relating specifically to bad genes will inhibit the expressionof these bad genes or the effects of the gene product of such bad genesand, thus, will provide yet another therapeutic mechanism in treatingthe effects of stroke, hypoxia and/or ischemia or other conditions inwhich apoptosis is to be inhibited and/or angiogenesis promoted.

[0181] x. Antibodies

[0182] The present invention also comprehends antibodies specific forthe proteins encoded by a naturally-occurring cDNA which is part of thepresent invention as discussed above. Such an antibody may be used fordiagnostic purposes to identify the presence of any suchnaturally-occurring proteins. Such antibody may be a polyclonal antibodyor a monoclonal antibody or any other molecule that incorporates theantigen-binding portion of a monoclonal antibody specific for such aprotein. Such other molecules may be a single-chain antibody, ahumanized antibody, an F(ab) fraction, a chimeric antibody, an antibodyto which is attached a label, such as fluorescent or radioactive label,or an immunotoxin in which a toxic molecule is bound to the antigenbinding portion of the antibody. The examples are intended to benon-limiting. However, as long as such a molecule includes theantigen-binding portion of the antibody, it will be expected to bind tothe protein and, thus, can be used for the same diagnostic purposes forwhich a monoclonal antibody can be used.

[0183] Conveniently, the antibodies can be prepared against theimmunogen or portion thereof for example a synthetic peptide based onthe sequence, or prepared recombinantly by cloning techniques or thenatural gene product and/or portions thereof can be isolated and used asthe immunogen. Immunogens can be used to produce antibodies by standardantibody production technology well known to those skilled in the art asdescribed generally in Harlow et al (1988) and Borrebaeck (1992).Antibody fragments can also be prepared from the antibodies and includeFab, F(ab′)₂, and Fv by methods known to those skilled in the art.

[0184] For producing polyclonal antibodies a host, such as a rabbit orgoat, is immunized with the immunogen or immunogen fragment, generallywith an adjuvant and, if necessary, coupled to a carrier; antibodies tothe immunogen are collected from the sera. Further, the polyclonalantibody can be absorbed such that it is monospecific. That is, the seracan be absorbed against related immunogens so that no cross-reactiveantibodies remain in the sera rendering it monospecific.

[0185] For producing monoclonal antibodies the technique involveshyperimmunization of an appropriate donor with the immunogen, generallya mouse, and isolation of splenic antibody producing cells. These cellsare fused to a cell having immortality, such as a myloma cell, toprovide a fused cell hybrid which has immortality and secretes therequired antibody. The cells are then cultured, in bulk, and themonoclonal antibodies harvested from the culture media for use.

[0186] For producing recombinant antibody (see generally Huston et al,1991; Johnson et al, 1991; Mernaugh et al, 1995), messenger RNAs fromantibody producing B-lymphocytes of animals, or hybridoma arereverse-transcribed to obtain complimentary DNAs (cDNAs). Antibody cDNA,which can be full or partial length, is amplified and cloned into aphage or a plasmid. The cDNA can be a partial length of heavy and lightchain cDNA, separated or connected by a linker. The antibody, orantibody fragment, is expressed using a suitable expression system toobtain recombinant antibody. Antibody cDNA can also be obtained byscreening pertinent expression libraries.

[0187] The antibody can be bound to a solid support substrate orconjugated with a detectable moiety or be both bound and conjugated, asis well known in the art. (For a general discussion of conjugation offluorescent or enzymatic moieties see Johnstone et al, 1982.) Thebinding of antibodies to a solid support substrate is also well known inthe art. (See for a general discussion Harlow et al, 1988, andBorrebaeck, 1992). The detectable moieties contemplated with S thepresent invention can include, but are not limited to, fluorescent,metallic, enzymatic and radioactive markers such as biotin, gold,ferritin, alkaline phosphatase, β-galactosidase, peroxidase, urease,fluorescein, rhodamine, tritium, ¹⁴C and iodination.

[0188] XI. Antisense Sequences

[0189] In order to manipulate the expression of a bad gene, it isdesirable to produce antisense RNA in a cell. To this end, the completeor partial cDNA of a bad gene in accordance with the present inventionis inserted into an expression vector comprising a promoter. The 3′ endof the cDNA is thereby inserted adjacent to the 3′ end of the promoter,with the 5′ end of the cDNA being separated from the 3′ end of thepromoter by said cDNA. Upon expression of the cDNA in a cell, anantisense RNA is therefore produced which is incapable of coding for theprotein. The presence of antisense RNA in the cell reduces theexpression of the cellular (genomic) copy of the bad gene.

[0190] For the production of antisense RNA, the complete cDNA may beused. Alternatively, a fragment thereof may be used, which is preferablybetween about 9 and 2,000 nucleotides in length, more preferably between15 and 500 nucleotides, and most preferably between 30 and 150nucleotides.

[0191] The fragment is preferably corresponding to a region within the5′ half of the cDNA, more preferably the 5′ region comprising the 5′untranslated region and/or the first exon region, and most preferablycomprising the ATG translation start site. Alternatively, the fragmentmay correspond to DNA sequence of the 5′ untranslated region only.

[0192] Antisense intervention in the expression of specific genes can beachieved by the use of synthetic AS oligonucleotide sequences (forrecent reports see Lefebvre-d'Hellencourt et al, 1995; Agrawal, 1996;Lev-Lehman et al, 1997). The oligonucleotide is preferably a DNAoligonucleotide. The length of the antisense oligonucleotide ispreferably between 9 and 150, more preferably between 12 and 60, andmost preferably between 15 and 50 nucleotides. Suitable antisenseoligonucleotides that inhibit the production of the protein of thepresent invention from its encoding mRNA can be readily determined withonly routine experimentation through the use of a series of overlappingoligonucleotides similar to a “gene walking” technique that iswell-known in the art. Such a “walking” technique as well-known in theart of antisense development can be done with synthetic oligonucleotidesto walk along the entire length of the sequence complementary to themRNA in segments on the order of 9 to 150 nucleotides in length. This“gene walking” technique will identify the oligonucleotides that arecomplementary to accessible regions on the target mRNA and exertinhibitory antisense activity.

[0193] The AS oligonucleotide sequence is designed to complement atarget mRNA of interest and form an RNA:AS duplex. This duplex formationcan prevent processing, splicing, transport or translation of therelevant mRNA. Moreover, certain AS nucleotide sequences can elicitcellular RNase H activity when hybridized with their target mRNA,resulting in mRNA degradation (Calabretta et al, 1996). In that case,RNase H will cleave the RNA component of the duplex and can potentiallyrelease the AS to further hybridize with additional molecules of thetarget RNA. An additional mode of action results from the interaction ofAS with genomic DNA to form a triple helix which can betranscriptionally inactive.

[0194] The sequence target segment for the antisense oligonucleotide isselected such that the sequence exhibits suitable energy relatedcharacteristics important for oligonucleotide duplex formation withtheir complementary templates, and shows a low potential forself-dimerization or self-complementation (Anazodo et al, 1996). Forexample, the computer program OLIGO 4.0 (National Biosciences, Inc.),can be used to determine antisense sequence melting temperature, freeenergy properties, and to estimate potential self-dimer formation andself-complimentary properties. The program allows the determination of aqualitative estimation of these two parameters (potential self-dimerformation and self-complimentary) and provides an indication of “nopotential” or “some potential” or “essentially complete potential”.Using this program target segments are generally selected that haveestimates of no potential in these parameters. However, segments can beused that have “some potential” in one of the categories. A balance ofthe parameters is used in the selection as is known in the art. Further,the oligonucleotides are also selected as needed so that analogsubstitution do not substantially affect function.

[0195] Alternatively, an oligonucleotide based on the coding sequence ofa protein capable of binding to a bad gene or the protein encodedthereby can be designed using Oligo 4.0 (National Biosciences, Inc.).Antisense molecules may also be designed to inhibit translation of anmRNA into a polypeptide by preparing an antisense which will bind in theregion spanning approximately −10 to +10 nucleotides at the 5′ end ofthe coding sequence.

[0196] The mechanism of action of antisense RNA and the current state ofthe art on use of antisense tools is reviewed in Kumar et al (1998).There are reviews on the chemical (Crooke, 1995; Uhlmann et al, 1990),cellular (Wagner, 1994) and therapeutic (Hanania, et al, 1995; Scanlon,et al, 1995; Gewirtz, 1993) aspects of this rapidly developingtechnology. The use of antisense oligonucleotides in inhibition of BMPreceptor synthesis has been described by Yeh et al (1998). The use ofantisense oligonucleotides for inhibiting the synthesis of thevoltage-dependent potassium channel gene Kvl.4 has been described byMeiri et al (1998). The use of antisense oligonucleotides for inhibitionof the synthesis of Bcl-x has been described by Kondo et al (1998). Thetherapeutic use of antisense drugs is discussed by Stix (1998); Flanagan(1998); Guinot et al (1998), and references therein. Within a relativelyshort time, ample information has accumulated about the in vitro use ofAS nucleotide sequences in cultured primary cells and cell lines as wellas for in vivo administration of such nucleotide sequences forsuppressing specific processes and changing body functions in atransient manner. Further, enough experience is now available in vitroand in vivo in animal models and human clinical trials to predict humanefficacy.

[0197] Modifications of oligonucleotides that enhance desired propertiesare generally used when designing antisense oligonucleotides. Forinstance, phosphorothioate bonds are used instead of the phosphoesterbonds that naturally occur in DNA, mainly because such phosphorothioateoligonucleotides are less prone to degradation by cellular enzymes. PengHo et al teach that undesired in vivo side effects of phosphorothioateoligonucleotides may be reduced when using a mixedphosphodiester-phosphorothioate backbone. Preferably,2′-methoxyribonucleotide modifications in 60% of the oligonucleotide isused. Such modified oligonucleotides are capable of eliciting anantisense effect comparable to the effect observed with phosphorothioateoligonucleotides. Peng Ho et al teach further that oligonucleotideanalogs incapable of supporting ribonuclease H activity are inactive.

[0198] Therefore, the preferred antisense oligonucleotide of the presentinvention has a mixed phosphodiester-phosphorothioate backbone.Preferably, 2′-methoxyribonucleotide modifications in about 30% to 80%,more preferably about 60%, of the oligonucleotide are used.

[0199] In the practice of the invention, antisense oligonucleotides orantisense RNA may be used. The length of the antisense RNA is preferablyfrom about 9 to about 3,000 nucleotides, more preferably from about 20to about 1,000 nucleotides, most preferably from about 50 to about 500nucleotides.

[0200] In order to be effective, the antisense oligonucleotides of thepresent invention must travel across cell membranes. In general,antisense oligonucleotides have the ability to cross cell membranes,apparently by uptake via specific receptors. As the antisenseoligonucleotides are single-stranded molecules, they are to a degreehydrophobic, which enhances passive diffusion through membranes.Modifications may he introduced to an antisense oligonucleotide toimprove its ability to cross membranes. For instance, theoligonucleotide molecule may be linked to a group which includespartially unsaturated aliphatic hydrocarbon chain and one or more polaror charged groups such as carboxylic acid groups, ester groups, andalcohol groups. Alternatively, oligonucleotides may be linked to peptidestructures, which are preferably membranotropic peptides. Such modifiedoligonucleotides penetrate membranes more easily, which is critical fortheir function and may, therefore, significantly enhance their activity.Palmityl-linked oligonucleotides have been described by Gerster et al(1998). Geraniol-linked oligonucleotides have been described by Shoji etal (1998). Oligonucleotides linked to peptides, e.g., membranotropicpeptides, and their preparation have been described by Soukchareun et al(1998). Modifications of antisense molecules or other drugs that targetthe molecule to certain cells and enhance uptake of the oligonucleotideby said cells are described by Wang (1998).

[0201] The antisense oligonucleotides of the invention are generallyprovided in the form of pharmaceutical compositions. These compositionsare for use by injection, topical administration, or oral uptake.

[0202] Preferred uses of the pharmaceutical compositions of theinvention by injection are subcutaneous injection, intraperitonealinjection, and intramuscular injection.

[0203] The pharmaceutical composition of the invention generallycomprises a buffering agent, an agent which adjusts the osmolaritythereof, and optionally, one or more carriers, excipients and/oradditives as known in the art, e.g., for the purposes of adding flavors,colors, lubrication, or the like to the pharmaceutical composition.

[0204] Carriers may include starch and derivatives thereof, celluloseand derivatives thereof, e.g., microcrystalline cellulose, Xanthum gum,and the like. Lubricants may include hydrogenated castor oil and thelike.

[0205] A preferred buffering agent is phosphate-buffered saline solution(PBS), which solution is also adjusted for osmolarity.

[0206] A preferred pharmaceutical formulation is one lacking a carrier.Such formulations are preferably used for administration by injection,including intravenous injection.

[0207] The preparation of pharmaceutical compositions is well known inthe art and has been described in many articles and textbooks, see e.g.,Remington's Pharmaceutical Sciences, especially pp 1521-1712 therein(Gennaro, 1990).

[0208] Additives may also be selected to enhance uptake of the antisenseoligonucleotide across cell membranes. Such agents are generally agentsthat will enhance cellular uptake of double-stranded DNA molecules. Forinstance, certain lipid molecules have been developed for this purpose,including the transfection reagents DOTAP (Boehringer Mannheim),Lipofectin, Lipofectam, and Transfectam, which are availablecommercially. For a comparison of various of these reagents in enhancingantisense oligonucleotide uptake, see e.g., Quattrone et al (1995) andCapaccioli et al (1993). The antisense oligonucleotide of the inventionmay also be enclosed within liposomes. The preparation and use ofliposomes, e.g., using the above-mentioned transfection reagents, iswell known in the art. Other methods of obtaining liposomes include theuse of Sendai virus or of other viruses. Examples of publicationsdisclosing oligonucleotide transfer into cells using the liposometechnique are, e.g., Meyer et al (1998), Kita et al (1999), Nakamura etal (1998), Abe et al (1998), Soni et al (1998), Bai et al (1998), seealso discussion in the same Journal p. 819-20, Bochot et al (1998),Noguchi et al (1998), Yang et al (1998), Kanamaru et al (1998), andreferences therein. The use of Lipofectin in liposome-mediatedoligonucleotide uptake is described in Sugawa et al (1998). The use offusogenic cationic-lipid-reconstituted influenza-virus envelopes(cationic virosomes) is described in Waelti et al (1998).

[0209] The above-mentioned cationic or non-ionic lipid agents not onlyserve to enhance uptake of oligonucleotides into cells, but also improvethe stability of oligonucleotides that have been taken up by the cell.

[0210] XII. Ribozymes

[0211] Instead of an antisense sequence as discussed herein above,ribozymes can be utilized. This is particularly necessary in cases whereantisense therapy is limited by stoichiometric considerations (Sarver etal, 1990). Ribozymes can then be used that will target the samesequence. Ribozymes are RNA molecules that possess RNA catalytic ability(see Cech for review) that cleave a specific site in a target RNA. Thenumber of RNA molecules that are cleaved by a ribozyme is greater thanthe number predicted by stochiochemistry. (Hampel et al, 1989;Uhlenbeck, 1987).

[0212] Given the known mRNA sequence of a gene, ribozymes, which are RNAmolecule that specifically bind and cleave said mRNA sequence (see,e.g., Chen et al (1992), Zhao et al (1993), Shore et al (1993), Josephet al (1993), Shimayama et al (1993), and Cantor et al (1993), may bedesigned.

[0213] Ribozymes catalyze the phosphodiester bond cleavage of RNA.Several ribozyme structural families have been identified includingGroup I introns, RNase P, the hepatitis delta virus ribozyme, hammerheadribozymes and the hairpin ribozyme originally derived from the negativestrand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan,1994; U.S. Pat. No. 5,225,347, columns 4-5). The latter two families arederived from viroids and virusoids, in which the ribozyme is believed toseparate monomers from oligomers created during rolling circlereplication (Symons, 1989 and 1992). Hammerhead and hairpin ribozymemotifs are most commonly adapted for trans-cleavage of mRNAs for genetherapy (Sullivan, 1994). The ribozyme type utilized in the presentinvention is selected as is known in the art. Hairpin ribozymes are nowin clinical trial and are the preferred type. In general the ribozyme isfrom 30-100 nucleotides in length.

[0214] Accordingly, a ribozyme-encoding RNA sequence may be designedthat cleaves the mRNA of a bad gene of the present invention. The siteof cleavage is preferably located in the coding region or in the 5′non-translated region, more preferably, in the 5′ part of the codingregion close to the AUG translational start codon.

[0215] A DNA encoding a ribozyme according to the present invention maybe introduced into cells by way of DNA uptake, uptake of modified DNA(see modifications for oligonucleotides and proteins that result inenhanced membrane permeability, as described above for oligonucleotidesand described below for proteins), or viral vector-mediated genetransfer.

[0216] XIII. Negative Dominant Peptides

[0217] Negative dominant peptide refers to a peptide encoded by a cDNAsequence that encodes only a part of a protein, i.e. a peptide (seeHerskowitz, 1987). This peptide can have a different function from theprotein it was derived from. It can interact with the full protein andinhibit its activity or it can interact with other proteins and inhibittheir activity in response to the full protein. Negative dominant meansthat the peptide is able to overcome the natural proteins and fullyinhibit their activity to give the cell a different characteristic, suchas resistance or sensitization to killing. For therapeutic interventioneither the peptide itself is delivered as the active ingredient of apharmaceutical composition or the cDNA can be delivered to the cellutilizing the same methods as for antisense delivery.

[0218] XIV. Introduction of Proteins, Peptides, and DNA into Cells

[0219] The present invention provides proteins encoded by good genes,peptides derived therefrom, antisense DNA molecules corresponding to badgenes, peptides which are negative dominant for bad genes, andoligonucleotides. A therapeutic or research-associated use of thesetools necessitates their introduction into cells of a living organism orinto cultured cells. For this purpose, it is desired to improve membranepermeability of peptides, proteins and oligonucleotides. Ways to improvemembrane permeability of oligonucleotides have been discussed above. Thesame principle, namely, derivatization with lipophilic structures, mayalso be used in creating peptides and proteins with enhanced membranepermeability. For instance, the sequence of a known membranotropicpeptide as noted above may be added to the sequence of the peptide orprotein. Further, the peptide or protein may be derivatized by partlylipophilic structures such as the above-noted hydrocarbon chains, whichare substituted with at least one polar or charged group. For example,lauroyl derivatives of peptides have been described by Muranishi et al(1991). Further modifications of peptides and proteins include theoxidation of methionine residues to thereby create sulfoxide groups, asdescribed by Zacharia et al (1991). Zacharia and coworkers alsodescribed peptide or derivatives wherein the relatively hydrophobicpeptide bond is replaced by its ketomethylene isoester (COCH₂). It isknown to those of skill in the art of protein and peptide chemistrythese and other modifications enhance membrane permeability.

[0220] Another way of enhancing membrane permeability is to make use ofreceptors, such as virus receptors, on cell surfaces in order to inducecellular uptake of the peptide or protein. This mechanism is usedfrequently by viruses, which bind specifically to certain cell surfacemolecules. Upon binding, the cell takes the virus up into its interior.The cell surface molecule is called a virus receptor. For instance, theintegrin molecules CAR and AdV have been described as virus receptorsfor Adenovirus (Hemmi et al, 1998, and references cited therein). TheCD4, GPR1, GPR15, and STRL33 molecules have been identified asreceptors/coreceptors for HIV (Edinger et al, 1998 and references citedtherein).

[0221] By conjugating peptides, proteins or oligonucleotides tomolecules that are known to bind to cell surface receptors, the membranepermeability of said peptides, proteins or oligonucleotides will beenhanced. Examples of suitable groups for forming conjugates are sugars,vitamins, hormones, cytokines, transferrin, asialoglycoprotein, and thelike molecules. Low et al U.S. Pat. No. 5,108,921 describes the use ofthese molecules for the purpose of enhancing membrane permeability ofpeptides, proteins and oligonucleotides, and the preparation of saidconjugates.

[0222] Low and coworkers further teach that molecules such as folate orbiotin may be used to target the conjugate to a multitude of cells in anorganism, because of the abundant and non-specific expression of thereceptors for these molecules.

[0223] The above use of cell surface proteins for enhancing membranepermeability of a peptide, protein or oligonucleotide of the inventionmay also be used in targeting the peptide, protein or oligonucleotide ofthe present invention to certain cell types or tissues. For instance, ifit is desired to target neural cells, it is preferable to use a cellsurface protein that is expressed more abundantly on the surface ofthose cells.

[0224] The protein, peptide or oligonucleotide of the invention maytherefore, using the above-described conjugation techniques, be targetedto a certain cell type. For instance, if it is desired to protect fromneurotoxic stress in neural cell, a good gene, or protein encodedthereby, or an antisense or ribozyme of the invention designed toinhibit a bad gene, may be targeted at such cells, for instance, byusing molecules that are expressed on these cells. The skilled personwill recognize the possibilities of using a cell surface marker selectedfrom a multitude of known markers of neural and other cells, and ofthese, further selecting those that are expressed constitutively orinducibly.

[0225] XV. Virus-Mediated Cellular Targeting

[0226] The proteins, peptides and antisense sequences of the presentinvention may be introduced into cells by the use of a viral vector. Theuse of a vaccinia vector for this purpose is described in Chapter 16 ofAusubel et al (1994-2000). The use of adenovirus vectors has beendescribed, e.g., by Teoh et al (1998), Narumi et al (1998), Pederson etal (1998), Guang-Lin et al (1998), and references therein, Nishida et al(1998), Schwarzenberger et al (1998), and Cao et al (1998). Retroviraltransfer of antisense sequences has been described by Daniel et al(1998). The use of SV-40 derived viral vectors and SV-40 based packagingsystems has been described by Fang et al (1997). The use ofpapovaviruses which specifically target B-lymphocytes, has beendescribed by Langner et al (1998).

[0227] When using viruses as vectors, the viral surface proteins aregenerally used to target the virus. As many viruses, such as the aboveadenovirus, are rather unspecific in their cellular tropism, it may bedesirable to impart further specificity by using a cell-type ortissue-specific promoter. Griscelli et al (1998) teach the use of theventricle-specific cardiac myosin light chain 2 promoter forheart-specific targeting of a gene whose transfer is mediated byadenovirus.

[0228] Alternatively, the viral vector may be engineered to express anadditional protein on its surface, or the surface protein of the viralvector may be changed to incorporate a desired peptide sequence. Theviral vector may thus be engineered to express one or more additionalepitopes which may be used to target said viral vector. For instance,cytokine epitopes, MHC class II-binding peptides, or epitopes derivedfrom homing molecules may be used to target the viral vector inaccordance with the teaching of the invention. The above Langer et al.(1998) reference teach the use of heterologous binding motifs to targetB-lymphotrophic papoaviruses.

[0229] XVI. Pharmaceutical Compositions

[0230] The pharmaceutical compositions of the invention are preparedgenerally as known in the art. Thus, pharmaceutical compositionscomprising nucleic acids, e.g., ribozymes, antisense RNA or antisenseoligonucleotides, are prepared as described above for pharmaceuticalcompositions comprising oligonucleotides and/or antisense RNA. The aboveconsiderations apply generally also to other pharmaceuticalcompositions. For instance, the pharmaceutical composition of theinvention may contain naked DNA, e.g., good genes or fragments orderivatives thereof and a pharmaceutically acceptable carrier as knownin the art. A variety of ways to enhance uptake of naked DNA is known inthe art. For instance, cationic liposomes (Yotsuyanagi et al, 1998),dicationic amphiphiles (Weissig et al, 1998), fusogenic liposomes(Mizuguchi et al, 1996), mixtures of stearyl-poly(L-lysine) and lowdensity lipoprotein (LDL), (terplex, Kim et al, 1998), and even wholebacteria of an attenuated mutant strain of Salmonella typhimurium(Paglia et al, 1998) have been used in the preparation of pharmaceuticalcompositions containing DNA.

[0231] Administration of virus particles has been described in prior artpublications, see, e.g., U.S. Pat. No. 5,882,877, where Adenovirus basedvectors and administration of the DNA thereof is described. The viralDNA was purified on a CsCl gradient and then dialyzed againstTris-buffered saline to remove CsCl. In these preparations, viral titers(pfu/ml) of 10¹⁴ to 10¹⁰ are preferably used. Administration of virusparticles as a solution in buffered saline, to be preferablyadministered by subcutaneous injection, is known from U.S. Pat. No.5,846,546. Croyle and coworkers (Croyle et al, 1998) describe a processfor the preparation of a pharmaceutical composition of recombinantadenoviral vectors for oral gene delivery, using CsCl gradients andlyophilization in a sucrose-containing buffer.

[0232] The active ingredients of the pharmaceutical composition caninclude oligonucleotides that are nuclease resistant needed for thepractice of the invention or a fragment thereof shown to have the sameeffect targeted against the appropriate sequence(s) and/or ribozymes.Combinations of active ingredients as disclosed in the present inventioncan be used including combinations of antisense sequences.

[0233] Where the pharmaceutical composition of the invention includes apeptide or protein according to the present invention, the compositionwill generally contain salts, preferably in physiological concentration,such as PBS (phosphate-buffered saline), or sodium chloride (0.9% w/v),and a buffering agent, such as phosphate buffer in water or in thewell-known PBS buffer. In the following section, the term “peptide” ismeant to include all proteins or peptides according to the invention.The preparation of pharmaceutical compositions is well known in the art,see e.g., U.S. Pat. Nos. 5,736,519, 5,733,877, 5,554,378, 5,439,688,5,418,219, 5,354,900, 5,298,246, 5,164,372, 4,900,549, 4,755,383,4,639,435, 4,457,917, and 4,064,236.

[0234] The peptide of the present invention, or a pharmacologicallyacceptable salt thereof is preferably mixed with an excipient, carrier,diluent, and optionally, a preservative or the like, pharmacologicallyacceptable vehicles as known in the art, see, e.g., the above U.S.patents. Examples of excipients include, glucose, mannitol, inositol,sucrose, lactose, fructose, starch, corn starch, microcrystallinecellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose,polyvinylpyrrolidone and the like. optionally, a thickener may be added,such as a natural gum, a cellulose derivative, an acrylic or vinylpolymer, or the like.

[0235] The pharmaceutical composition is provided in solid, liquid orsemi-solid form. A solid preparation may be prepared by blending theabove components to provide a powdery composition. Alternatively, thepharmaceutical composition is provided as a lyophilized preparation. Theliquid preparation is provided preferably as an aqueous solution,aqueous suspension, oil suspension or microcapsule composition. Asemi-solid composition is provided preferably as hydrous or oily gel orointment. About 0.001 to 60 w/v %, preferably about 0.05 to 25 w/v % ofpeptide is provided in the composition.

[0236] A solid composition may be prepared by mixing an excipient with asolution of the peptide of the invention, gradually adding a smallquantity of water, and kneading the mixture. After drying, preferably invacuo, the mixture is pulverized. A liquid composition may be preparedby dissolving, suspending or emulsifying the peptide of the invention inwater, a buffer solution or the like. An oil suspension may be preparedby suspending or emulsifying the peptide of the invention or protein inan oleaginous base, such as sesame oil, olive oil, corn oil, soybeanoil, cottonseed oil, peanut oil, lanolin, petroleum jelly, paraffin,Isopar, silicone oil, fatty acids of 6 to 30 carbon atoms or thecorresponding glycerol or alcohol esters. Buffers include Sorensenbuffer (Ergeb Physiol, 12:393, 1912), Clark-Lubs buffer (J Bact, 2(1):109, 191, 1917), Macllvaine buffer (J Biol Chem, 49:183, 1921),Michaelis buffer (Die Wasserstoffinonenkonzentration, p. 186, 1914), andKolthoff buffer (Biochem Z, 179:410, 1926).

[0237] A composition may be prepared as a hydrous gel, e.g., fortransnasal administration. A hydrous gel base is dissolved or dispersedin aqueous solution containing a buffer, and the peptide of theinvention, and the solution warmed or cooled to give a stable gel.

[0238] Preferably, the peptide of the invention is administered throughintravenous, intramuscular or subcutaneous administration. Oraladministration is expected to be less effective, because the peptide maybe digested before being taken up. Of course, this consideration mayapply less to a peptide of the invention which is modified, e.g., bybeing a cyclic peptide, by containing non-naturally occurring aminoacids, such as D-amino acids, or other modifications which enhance theresistance of the peptide to biodegradation. Decomposition in thedigestive tract may be lessened by use of certain compositions, forinstance, by confining the peptide of the invention in microcapsulessuch as liposomes. The pharmaceutical composition of the invention mayalso be administered to other mucous membranes. The pharmaceuticalcomposition is then provided in the form of a suppository, nasal sprayor sublingual tablet. The dosage of the peptide of the invention maydepend upon the condition to be treated, the patient's age, bodyweight,and the route of administration, and will be determined by the attendingphysician.

[0239] The uptake of a peptide of the invention may be facilitated by anumber of methods. For instance, a non-toxic derivative of the choleratoxin B subunit, or of the structurally related subunit B of theheal-labile enterotoxin of enterotoxic Eschericia coli may be added tothe composition, see U.S. Pat. No. 5,554,378.

[0240] In another embodiment, the peptide of the invention is providedin a pharmaceutical composition comprising a biodegradable polymerselected from poly-1,4-butylene succinate, poly-2,3-butylene succinate,poly-1,4-butylene fumarate and poly-2,3-butylene succinate,incorporating the peptide of the invention as the pamoate, tannate,stearate or palmitate thereof. Such compositions are described, e.g., inU.S. Pat. No. 5,439,688.

[0241] In a further embodiment, a composition of the invention is a fatemulsion. The fat emulsion may be prepared by adding to a fat or oilabout 0.1-2.4 w/w of emulsifier such as a phospholipid, an emulsifyingaid, a stabilizer, mixing mechanically, aided by heating and/or removingsolvents, adding water and isotonic agent, and optionally, adjustingadding the pH agent, isotonic agent. The mixture is then homogenized.Preferably, such fat emulsions contain an electric charge adjustingagent, such as acidic phospholipids, fatty acids, bilic acids, and saltstherof. Acidic phospholipids include phosphatidylserine,phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. Bilicacids include deoxycholic acid, and taurocholic acid. The preparation ofsuch pharmaceutical compositions is described in U.S. Pat. No.5,733,877.

[0242] The pharmaceutical compositions containing the active ingredientsof the present invention as described herein above are administered anddosed in accordance with good medical practice, taking into account theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the medical arts. Theamount must be effective to achieve improvement including but notlimited to improved survival rate or more rapid recovery, or improvementor elimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the medical arts. Thepharmaceutical compositions can be combinations of the activeingredients but will include at least one active ingredient.

[0243] The doses can be single doses or multiple doses over a period ofseveral days. The treatment generally has a length proportional to thelength of the disease process and drug effectiveness and the patientspecies being treated.

[0244] In one embodiment, the compound of the present invention can beadministered initially by intravenous injection to bring blood levels toa suitable level. The patient's levels are then maintained by an oraldosage form, although other forms of administration, dependent upon thepatient's condition and as indicated above, can be used. The quantity tobe administered will vary for the patient being treated and will varyfrom about 100 ng/kg of body weight to 100 mg/kg of body weight per dayand preferably will be from 10 μg/kg to 10 mg/kg per day.

[0245] XVII. Knock-Out or Transgenic Animals

[0246] Transgenic Mice

[0247] The introduction of gene constructs into the genome of mice(transgenic mice) is a well-established procedure. Transgenic miceprovide the opportunity to examine the phenotypic outcome ofover-expression or ectopic expression of genes (gain-of-functionexperiments). Specific phenotypes obtained after such expression is avery strong predictor of gene function. Many human genes have beenexpressed in transgenic mice and in most cases they functionappropriately. Thus ,for the purpose of examining gain-of-function,human genes can be used. Specific plasmid vector constructs areavailable. They carry any of a variety of promoters that allowexpression of the gene in specific tissues. For example, promoters thatare brain specific are available, liver specific promoters,vascular-endothelial cell specific promoters, bone specific promoters,cardiac muscle specific promoters and many more. While mice arespecifically discussed herein as the transgenic animal, those ofordinary skill in the art well understand that any other eukaryoticanimal can be used in the same way as described for mice to make acorresponding transgenic animal.

[0248] Knockout Mice

[0249] Loss-of-function experiments in mice are mostly done by thetechnique of gene knockout. The technology is well established. Itrequires the use of mouse genes for the purpose of generating knockoutof the specific gene in embryonic stem (ES) cells that are thenincorporated into the mouse germ-line cells from which mice carrying thegene knockout are generated. From a human gene there are several ways torecover the homologous mouse gene. One way is to use the human gene toprobe mouse genomic libraries of lambda phages, cosmids or sACs.Positive clones are examined and sequenced to verify the identity of themouse gene. Another way is to mine the mouse EST database to find thematching mouse sequences. This can be the basis for generatingprimer-pairs or specific mouse probes that allow an efficient screen ofthe mouse genomic libraries mentioned above by PCR or by hybridization.For the vast majority of genes the mouse homologue of the human generetains the same biological function. The loss-of-function experimentsin mice indicate the consequences of absence of expression of the geneon the phenotype of the mouse and the information obtained is applicableto the function of the gene in humans. On many occasions a specificphenotype observed in knockout mice was similar to a specific humaninherited disease and the gene was then proved to be involved andmutated in the human disease. While mice are specifically discussedherein as the knockout animal, those of ordinary skill in the art wellunderstand that any other eukaryotic animal can be used in the same wayas described for mice to make a corresponding knockout animal.

[0250] The transgenics and knock-outs of the present invention areconstructed using standard methods known in the art and as set forth inU.S. Pat. Nos. 5,487,992, 5,464,764, 5,387,742, 5,360,735, 5,347,075,5,298,422, 5,288,846, 5,221,778, 5,175,385, 5,175,384, 5,175,383,4,736,866 as well as Burke et al (1991), Capecchi (1989), Davies et al(1992), Dickinson et al (1993), Duff et al (1995), Huxley et al (1991),Jakobovits et al (1993), Lamb et al (1993), Pearson et al (1993),Rothstein (1991), Schedl et al (1993), Strauss et al (1993). Further,patent applications WO 94/23049, WO 93/14200, WO 94/06908, WO 94/28123also provide information.

[0251] More specifically, any techniques known in the art can be used tointroduce the transgene expressibly into animals to produce the parentallines of animals. Such techniques include, but are not limited to,pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus mediatedgene transfer into germ lines (Van der Putten et al, 1985); genetargeting in embryonic stem cells (Thompson et al, 1989; Mansour, 1990and U.S. Pat. No. 5,614,396); electroporation of embryos (Lo, 1983); andsperm-mediated gene transfer (Lavitrano et al, 1989). For a review ofsuch techniques see Gordon (1989).

[0252] Further, one parent strain instead of carrying a direct humantransgene can have the homologous endogenous gene modified by genetargeting such that it approximates the transgene. That is, theendogenous gene has been “humanized” and/or mutated (Reaume et al,1996). It should be noted that if the animal and human sequence areessentially homologous a “humanized” gene is not required. Thetransgenic parent can also carry an over expressed sequence, either thenon-mutant or a mutant sequence and humanized or not as required. Theterm transgene is therefore used to refer to all these possibilities.

[0253] Additionally, cells can be isolated from the offspring whichcarry a transgene from each transgenic parent and that are used toestablish primary cell cultures or cell lines as is known in the art.

[0254] Where appropriate, a parent strain will be homozygous for thetransgene. Additionally, where appropriate, the endogenous non-transgenein the genome that is homologous to the transgene will benon-expressive. By non-expressive is meant that the endogenous gene willnot be expressed and that this non-expression is heritable in theoffspring. For example, the endogenous homologous gene could be“knocked-out” by methods known in the art. Alternatively, the parentalstrain that receives one of the transgenes could carry a mutation at theendogenous homologous gene rendering it non-expressed.

[0255] XVIII. Promoters

[0256] As promoters and regulatory elements of the candidate genes inaccordance with the present invention are also useful in the screeningassays described in Section VIII, the present invention is also directedto the sequence of such promoters and/or other regulatory agents. Oncethe gene has been identified, it is within the routine skill in the artfor one ordinary skill to identify the sequence of the promoter regionor other regulatory regions. This may be accomplished as discussedbelow.

[0257] It is well recognized that promoters are generally locatedupstream of the coding sequence. There are numerous methods usedconventionally in the art for determining a promoter region and portionsof that region essential for promoter activity. For example, Kahari etal (1990) made constructs in which a region from −2260 to −14 upstreamof the ATG initiation codon of the human elastin gene was systematicallytruncated from −2260 towards −14 to create a set of nested deletions,all with the same −14 end point, which is linked to and controls theexpression of a coding sequence for a reporter molecule (chloramphenicolacetyltransferase). The constructs are assayed for the expression of thereporter as a measure of the promoter activity of the truncated DNAfragments. Using this type of deletion analysis, Kahari et al isolated a497 bp fragment which provided maximal gene expression.

[0258] The above method is directed to locating the promoter region, aswell as identifying the portions thereof essential for activity. Othermutagenesis techniques, such as linker scanning, which generate a seriesof clustered point mutations can also be used to fine map the sequenceelements required for promoter function.

[0259] Although in a great majority of cases the 5′-flanking region issufficient to promote gene expression, it has been reported that in someinstances intron, or even the 3′-untranslated sequences, provideregulatory sequences that contribute to promoter activity. For example,intron I sequences were found to be important for high-level andtissue-specific expression of an alpha-skeletal actin gene, abeta-globin gene and a peripherin gene (Reecy et al, 1998;James-Pederson et al, 1995; Belecky-Adams et al, 1993). In view of theseexamples of introns or 31-untranslated sequences contributing topromoter activity, promoter constructs (i.e., fused to reporter gene)may include intron I sequences of the candidate gene and, whennecessary, 3′-untranslated sequences. In the former case, a DNA fragmentcan be isolated that spans the 5′-flanking region, the first exon andthe first intron, followed by the reporter gene. The translationinitiation codon of the candidate gene could also be mutated to avoidtranslation of truncated candidate gene product.

[0260] XIX. Examples

[0261] General Methods

[0262] Most of the techniques used in molecular biology are widelypracticed in the art, and most practitioners are familiar with thestandard resource materials which describe specific conditions andprocedures. However, for convenience, the following paragraphs can serveas a guideline.

[0263] General methods in molecular biology: Standard molecular biologytechniques known in the art and not specifically described weregenerally followed as in Sambrook et al (1989), and in Ausubel et al(1989), particularly for the Northern Analysis and in situ analysis andin Perbal (1988), and in Watson et al. Polymerase chain reaction (PCR)was carried out generally as in PCR Protocols: A Guide To Methods AndApplications, Academic Press, San Diego, Calif. (1990).

[0264] Reactions and manipulations involving other nucleic acidtechniques, unless stated otherwise, were performed as generallydescribed in Sambrook et al (1989), and methodology as set forth in U.S.Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference.

[0265] Additionally, in situ (In cell) PCR in combination with flowcytometry can be used for detection of cells containing specific DNA andmRNA sequences (Testoni et al, 1996).

[0266] General methods in immunology: Standard methods in immunologyknown in the art and not specifically described are generally followedas in Stites et al (1994) and Mishell et al (1980). Availableimmunoassays are extensively described in the patent and scientificliterature. See, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521 as well as Sambrook et al (1989).

[0267] General Methods of the Invention

[0268] The general methods of the invention are generally as describedin U.S. patent application Ser. No. 09/309,862 of same applicant whichis by reference incorporated herein in its entirety.

[0269] In brief, U.S. Ser. No. 09/309,862 provides methods foridentifying genes regulated at the RNA level by cue-induced geneexpression. It relates to the rapid isolation of differentiallyexpressed or developmentally regulated gene sequences through analysisof mRNAs obtained from specific cellular compartments and comparing thechanges in the relative abundance of the mRNA in these compartments as aresult of applying a cue to the tested biological samples. The cellularcompartments include polysomal and non-polysomal fractions, nuclearfractions, cytoplasmic fractions and splicesomal fractions. The methodincludes the steps of exposing cells or tissue to a cue or stimulus suchas mechanical, chemical, toxic, pharmaceutical or other stress,hormones, physiological disorders or disease; fractionating the cellsinto compartments such as polysomes, nuclei, cytoplasm and splicesomes;extracting the mRNA from these fractions, and subjecting the mRNA todifferential analysis using accepted methodologies, such as geneexpression array (GEM).

[0270] The method is designed for identifying and cloning genes whichare either up- or down-regulated responsive to a specific pathology,stress, physiological condition, and so on, and in general, to anyfactor that can influence cells or organisms to alter their geneexpression.

[0271] Further in U.S. Ser. No. 09/309,862, an example is provided whichshows the use of RNA isolation from nuclei for isolating genes whosesteady state levels show only minor changes, but which show highdifferential expression when detected by nuclear RNA probe. Most suchgenes are regulated at the transcriptional level.

[0272] The specific mRNA of the invention is total cellular mRNA, andregulation is specifically on the transcriptional level.

[0273] In order to identify genes whose expression is either induced orreduced by hypoxia, the following experimental techniques wereconducted.

[0274] Preparation of Custom Hypoxia-Specific Microarrays

[0275] The first step in identifying the genes of the present inventioninvolves the preparation of a microarray containing genes which aresuspected of either being induced by hypoxia after 16 hours, reduced byhypoxia after 16 hours, or induced by hypoxia after 4 hours, which genesare obtained either from the rat C6 glioma cell line or the human A172glioma cell line.

[0276] In the preparation of such a microarray, each of the cell lineswere exposed to hypoxia conditions (0.5% O₂ and 5% CO₂) for 4 or 16hours and compared to cells grown under normal conditions (normoxia).Three enriched libraries were made by the suppression subtractivehybridization (SSH) method using the “PCR-Select cDNA subtraction kit”from CLONTECH. The subtractive libraries were made from the followingsample:

[0277] 1. 16 hours hypoxia vs. normal (genes induced by hypoxia after 16hours).

[0278] 2. normal vs. 16 hours hypoxia (genes reduced by hypoxia after 16hours).

[0279] 3. 4 hours hypoxia vs. normal (genes induced by hypoxia after 4hours).

[0280] From library 1, 1000 colonies were grown, and the plasmidsprepared in 96 well format. From libraries 2 and 3, 500 colonies wereprocessed from each. Thus, a total of 2000 individual plasmids wereprepared and used for the fabrication of a Gene Expression Microarray(GEM). For this, the inserts of each plasmid were amplified by PCR androbotically fabricated on the glass. CDNA chip printing was performed bySynteni (Wang et al, 1999)

[0281] Preparation of Probes for Microarray Hybridization

[0282] Isolated messenger RNA is labeled with fluorescent dNTP's using areverse transcription reaction, using 50 μg template RNA (probes derivedfrom nuclear RNA and total RNA), to generate a labeled cDNA probe. mRNAis extracted from either C6 or A172 cells cultured in normoxiaconditions and labeled with Cy3-dCTP (Amersham) and mRNA extracted fromC6 or A172 cells cultured under hypoxic conditions is labeled withCy5-dCTP (Amersham). The two labeled cDNA probes are then mixed andhybridized onto microarrays (Schena et al, 1996; Wang et al, 1999).Following hybridization the microarray was scanned using a laser scannerand the amount of fluorescence of each of the fluorescence dyes wasmeasured for each cDNA clone on the microarray giving an indication ofthe level of mRNA in each of the original mRNA populations being tested.Comparison of the fluorescence on each cDNA clone on the microarraybetween the two different fluorescent dyes is a measure for thedifferential expression of the indicated genes between the twoexperimental conditions.

[0283] The following probes were made from C6 and A172 for screening theGEM:

[0284] 1. Normoxia (Cy3 labeled)+16 hours hypoxia (Cy5 labeled).

[0285] 2. Normoxia (Cy3 labeled)+4 hours hypoxia (Cy5 labeled).

[0286] The following CDNA sequences of the present invention were foundto be induced under hypoxic conditions.

[0287] In Situ Analysis:

[0288] In situ analysis is performed for the candidate genes identifiedby the differential response to exposure to hypoxic conditions asdescribed above. The expression is studied in normal tissues and inpathological models as described herein.

[0289] Utilizing microarray hybridization the sequences set forth hereinwere identified and cloned as being differentially expressed underhypoxic conditions (see also Braren et al, 1997).

[0290] In parallel experiments Northern Analysis results and resultsobtained by the gene expression microarray analysis where found tocoincide and either can be used to determine hypoxia-regulated response.As well in other experiments, the results from in situ analysis showed ahigh degree of correlation with the Northern Analysis and microarrayanalysis.

[0291] The sequences are listed that were found, the sequences areidentified by clone number. In some cases either end of the clone hasbeen sequenced for use or the entire clone sequence and protein sequenceare provided.

[0292] Unigem1 (Syntheni) was utilized for screening of human gliomacell line A172 to identify genes whose expression is modified byhypoxia.

[0293] A Retinopathy Model

[0294] Three major biological processes occur in nervous tissues underhypoxic conditions:

[0295] 1. apoptotic death of hypoxia-damaged cells;

[0296] 2. angiogenesis induced by factors secreted by hypoxia-sufferingcells (a feedback control of oxygen concentration in tissue); and

[0297] 3. secretion of neurotrophic and neuroprotective factors.

[0298] Therefore, it was assumed that among novel genestranscriptionally regulated by hypoxia in C6 and A172 glioma cells,there are those with pro- and antiapoptotic function as well as secretedneurotrophic, neuroprotective and angiogenic factors. It is worthnoting, that regulation of apoptosis and angiogenesis is closely linkedto cancerogenesis.

[0299] As initial step of biological characterization, candidate geneswere tested for their ability to induce/protect cells from apoptosis,for neurotrophic activity and for angiogenic/antiangiogenic activity.

[0300] Cell Culture

[0301] MCF7 Tet-off (Clontech) human epithelial breast carcinoma cellsand their transfected derivatives were maintained in DMEM supplementedwith 10% FCS, 2 mM L-glutamine, 20 U/ml penicillin, 20 μg/mlstreptomycin, and 100 μg/ml neomycin. The transfectants were cultured inthe presence of 1 μg of tetracycline per ml. For UV treatment, cellswere irradiated with 100 Mj/cm² short wavelength UV (UV Crosslinker,Fisher) and then incubated at 37° C. for 24 hours. Cells were stainedwith 0.5% methylene blue in 50% ethanol.

[0302] Human umbilical vein endothelial cells (HUVEC) were grown in M199medium supplemented with 20% FCS, 2 mM L-glutamine, 20 U/ml penicillin,20 μg/ml streptomycin, 0.001 mg/ml Heparin, 0.1 mg/ml ECGS.

[0303] Expression Vectors and Transfection Methods

[0304] pTet-Splice/95 flag construct was prepared by EcoRI/HindIIIsubcloning from pLPC flag into pTet-splice.

[0305] MCF7 Tet-off cells were transfected with pTet-Splice/95 Flagusing lipofectamine reagent. Stable transfectants were obtained bycotranfection of 0.5 μg of a thymidine kinase hygromycin plasmid. Cellswere selected with 100 μg per ml hygromycin in the presence of 2 μg perml tetracycline in the medium. Clones were screened fortetracycline-sensitive HP95 expression by Northern blot.

[0306] Growth Rate Analysis

[0307] MCF7 cells and their HP95 transfectants were seeded at 10⁴ cellsper 35-mm-diameter dish with or without tetracycline. At dailyintervals, cells were collected by trypsinization and counted. Thisexperiment was done in triplicate.

[0308] Assessment of Cell Viability

[0309] The cell viability was estimated by the lactate dehydrogenase(LDH) leakage method using a Cytotoxicity Detection Kit (MolecularBiochemicals) according to the manufacturer's protocol. LDH activity wasmeasured as the optimal density at 492 nm.

[0310] Ischemia

[0311] Ischemia was achieved by incubating cells in a glucose freemedium in a humidified environment at 37° C. in a three gas incubatormaintained at 5% CO₂ and 0.5% O₂ for 16 hours.

[0312] Oxidative Stress

[0313] MCF7 cells were treated by adding to complete medium freshlyprepared hydrogen peroxide at the concentration of 1mM for 24 hours.

[0314] Serum starvation Experiment

[0315] MCF7 clones were plated at 10⁴ cells in six-well plates in DMEMcontaining 10% FCS with or without tetracycline. The medium was replaced72 hours later with medium containing 0.1% serum in the presence orabsence of tetracycline. After 24 hours cell viability was measured.

[0316] Annexin V Apoptosis Assay

[0317] The MCF7 clones were seeded into 60 mm culture dishes (1×10⁵cells/dish) and were maintained in the presence or absence oftetracycline for 72 hours. The cells were collected by trypsinization,centrifuged and washed in phosphate-buffered saline (PBS). The cellswere then resuspended in 200 μl of 1× binding buffer. The apoptoticcells were analyzed using a Annexin V apoptosis assay kit (ALEXISBiochemicals) according to the manufacturer's protocol.

[0318] Western Blot Analysis

[0319] Cells were washed with phosphate-buffered saline, and lysed inlysis buffer containing 10 mM Tris-HCl, pH 7.4, 1% (v/v) Noidet P40,0.1% (w/v) sodium deoxycholate, 0.1% (w/v) sodium deoxycholate, 0.1%(w/v) SDS, 0.15 M NaCl, and protease inhibitor cocktail (BoehringerMannheim). The whole cell lysates were clarified by centrifugation at12,000 x g for 30 minutes. Lysates containing 30 μg of protein werefractionated by SDS- 10% polyacrylamide gel and transferred ontomembrane (Schleicher & Schuell). The blots were incubated with antibodyspecific for Bcl-2 (Transduction Laboratories) and with the secondantibody for detection of Bcl-2 using the ECL detection system(Amersham). Collection of Conditioned Medium

[0320] The MCF7 clones (1×10⁵ cells/dish) were grown in HUVEC medium inthe presence or absence of tetracycline for 72 hours. Cell-conditionedmedia were collected, centrifuged at 15,000×g for 10 minutes. HUVEC,MCF7 and PC12 cells were seeded into 6 wells culture dishes (3×10⁴cells/well) 72 hours later, the conditioned medium was added (1:1).After 24 hours cell viability was measured.

[0321] Middle Cerebral Artery Occlusion (MCAO) Stroke Model

[0322] The stroke model was implied in the stroke-prone spontaneouslyhypertensive rat strain. Occlusion was permanent and unilateral—byelectrocoagulation of MCA. This led to focal brain ischemia at theipsilateral side of brain cortex leaving the contralateral side intact(control). Experimental animals were sacrificed 1, 2, 4, 12, 24, 48 and72 hours after the operation, respectively. Brains were removed, fixedin formalin, embedded into paraffin and coronal sections were performedfor the further use in in situ hybridization with Hypoptin-specificriboprobes. VEGF- and PGK-specific rihoprobes were used as positivecontrols.

[0323] In Situ Hybridization

[0324] Radioactive in situ hybridization was performed according topreviously published protocol (Faerman et al, 1997) with slightmodifications. Deparaffinized sections were heated in 2×SSC at 70° C.for 30 minutes, rinsed in distilled water and incubated with 10 mg/mlproteinase K in 0.2M Tris-HCl (pH7.4), 0.05 M EDTA at 37° C. for 20minutes. After proteinase digestion, slides were postfixed in 4%paraformaldehyde in PBS (20 minutes), quenched in 0.2% glycine (5minutes), rinsed in distilled water, rapidly dehydrated through gradedethanols and air-dried. The hybridization mixture contained 50%formamide, 4×SSC (pH 8.0), 1× Denhardt's, 0.5 mg/ml herring sperm DNA,0.25 mg/ml yeast RNA, 10 mM DTT, 10% dextran sulfate and 2×10⁴ cpm/μl of[³⁵S]-UTP-labeled riboprobe. After application of the hybridizationmixture, sections were covered with sheets of polypropylene film cutfrom autoclavable disposposal bags and incubated in humidified chamberat 65° C. overnight. After hybridization covering film was floated offin 5×SSC with 10 mM DTT at 65° C. and slides were washed at highstringency: 2XSSC, 50% formamide, 10 mM DTT at 65° C. for 30 minutes andtreated with RNAse A (10 μg/ml) for 30 minutes at 37° C. The highstringency washing step was repeated and slides were next washed in2×SSC and 0.1×SSC (15 minutes each) at 37° C. Then slides were rapidlydehydrated through ascending ethanols and air-dried. For autoradiographyslides were dipped in Kodak NTB-2 nuclear track emulsion diluted 1:1with double-distilled water and were exposed for 3 weeks in light-tightbox containing dessicant at 4° C. Exposed slides were developed in KodakD-19 developer, fixed in Kodak fixer and counterstained withhematoxilin-eosin.

[0325] Microphotographs were taken using Zeiss Axioscop-2 microscopeequipped with Diagnostic Instruments Spot RT CCD camera.

[0326] The sequences of the invention, the methods used therewith andthe utility of the present invention can be shown by the followingnon-limiting examples:

EXAMPLE 1 92 SEQ ID NO: 1

[0327] Northern Blot Analysis:

[0328] Gene 92 is found up-regulated after 16 hours of hypoxia. OnNorthern blots, it appears as a single 5 Kb transcript.

[0329] Cloning:

[0330] Several partial human cDNA clones corresponding to gene 92 wereisolated from human A172 cDNA library. The length of available contig is2212 bp and it contains an ORF potentially coding for a 437 amino acid(265-1576 bp) protein (SEQ ID NO: 2). The putative initiating ATO codonis preceded by in frame stop codon.

[0331] Bioinformatic Analysis:

[0332] Similarity search with 92 cDNA sequence against the publicdatabases have shown 60% similarity to unknown Drosophila DNA sequence(AC004283) and mainly encompasses the 3′ UTR and a part of the codingsequence. The search against the protein public databases gave partialsimilarity to hypothetical C. elegans protein (1703624) (77% similarityand 46% Identity).

[0333] The 92 cDNA sequence contains a region of 55 nucleotides (336-390bp) that is constituted of CGG repeats. On the level of amino acids itappears as a GGD/SFGG (SEQ ID NO: 20) repeated unit (aa 24-44). Two ofthe isolated cDNA clones contain a 30 nucleotides in frame deletionwithin this region, indicating that the amount of repeats can bevariable. Forty-four of these nucleotides form a strong stem and loopsecondary structure. When 92 cDNA was in vitro translated, the obtainedprotein had much smaller size than expected (30 kD instead of 45 kD).This, means that the stem and loop structure formed right downstream tothe putative initiation codon prevents the proper progression ofribosome and the initiation actually starts from the next in frame ATGlocated at position 820-822.

EXAMPLE 2 95 SEQ ID NO: 3

[0334] Identification of Gene 95 mRNA Induction under Hypoxic Treatments

[0335] CDNA microarray differential expression was performed in order toidentify genes that were responsive to hypoxia in human A172 gliomacells. 95 mRNA levels were significantly elevated under hypoxia.Northern blot analysis was performed in order to verify theseobservations. The 95 mRNA levels (3.9 kb) were highly induced by hypoxiain A172 glioma cells. Human EST that contained a full-length cDNA wasidentified as the human 95 transcript (SEQ ID NO: 3) . By in vitrotranslation this cDNA gave rise to a protein product of 62 Kd (SEQ IDNO: 4) . The sequence is 480 aa corresponding to nucleotides 323-1762 ofSEQ ID NO: 3.

[0336] Gene 95 shares homology with the PA26 gene (FIG. 1). However,PA26 mRNA levels were not influenced by hypoxia in A172 cells (resultsnot shown). Incubation of various cell lines from different origin(H1299, MCF7, Skov3) revealed high induction of 95 mRNA after 4 and 16hours of hypoxic treatments (results not shown). p53 was not essentialfor the hypoxia-induced up-regulation, since 95 mRNA levels wereincreased during hypoxia, regardless of the p53 status of the cells.

[0337] The results from testing on a variety of cell lines prove thatthe hypoxia-induced up-regulation of expression of this gene is notlimited to a specific cell line, but is found in a variety of celllines. This confirms the expectation that such up-regulation will befound in any human cell subjected to hypoxia. Therefore, gene 95 and itsencoded protein are excellent candidates for diagnostic testing oftissue or fluids for having been subjected to hypoxia, as describedabove.

[0338] It is known that HIF-1 mediated gene transactivation involvesdistinct nucleic acid motifs, namely HREs, in its binding. Accordingly,computer software from Genomatix GmbH was used to analyze the human 95gene for appropriate HRE sequences. In this analysis, the HRE consensusmotif proposed by Wenger et al (1997) was followed. Two putative HREsites were found in intron 1 and intron 2 of 95 gene (not shown),suggesting an HIF-1-dependent regulation of 95 gene transcription.

[0339] Gene 95 mRNA Was Up-Regulated Following DNA Damage in a p53Dependent Manner

[0340] The effect of DNA damage on 95 mRNA was examined. Different celllines were exposed to doxorubicin, a DNA-damaging agent that induces DNAbreaks, or to UV radiation. 95 mRNA was strongly induced 24 hours afterdoxorubicin treatment in p53 wild type cells (MCF7, HEF and 293). Incontrast, no induction was detected in p53-deficient cells (MDAH041,H1299). Similar results were obtained for cells exposed to UV radiation(data not shown). To verify the hypothesis on regulation of 95 by p53under DNA-damage, MCF7 and their derivatives transduced with GSE56 (p53dominant negative) were exposed to doxorubicin, UV radiation andhypoxia. GSE56 completely abrogated the induction of 95 by DNA-damagingagents, but did not affect its induction by hypoxia.

[0341] Inducible Expression of 95 in MCF7 Cells Revealed Delay in TheirGrowth Rate and induced Apoptosis

[0342] To permit conditional expression of a potential antiproliferativegene, human epithelial breast carcinoma MCF7 cells were stablytransfected with a tetracycline-repressible vector containingflag-epitope-tagged 95. Two clones of MCF7 cells, which showedtetracycline-sensitive expression of 95 were obtained by Northern blot.To investigate how 95 overexpression affects the growth rate ofproliferating breast tumor cells, the growth of the transfectant clonesand control clones in the presence or absence of tetracycline wasdetermined. As shown in FIG. 2, 95 overexpressing clones showedsignificant delay in growth compared with non-induced cells.

[0343] In order to determine whether this growth inhibition was due to95-induced cell apoptosis, the 95 inducible clones were grown for 72hours in the presence or absence of tetracycline. Overexpression of 95resulted in cell apoptosis as determined by Annexin V apoptosis assay.Since it is known that Bcl-2 has a protective effect against apoptosis,its expression in MCF7-95 induced clones by Western analysis was tested.Dephosphorylated-Bcl-2 expression was induced in 95 overexpressingclones.

[0344] 95 Induced DNA Damaged Apoptosis in MCF7 Cells

[0345] To find whether DNA damaged agents can stimulate apoptosis in 95overexpressing cells, MCF7-95 inducible clones were treated withdoxorubicin or exposed to UV irradiation in the presence or absence oftetracycline. Both stimuli induced apoptosis in >90% of the MCF7-95expressing cells. Treatment with taxol, which is an antimicrotubuleagent, revealed no difference between MCF7-95 inducible and controlclones.

[0346] To investigate how 95 overexpression affects the response ofproliferating breast tumor cells to mitogens, the response toserum-starved conditions (0.1% serum) was determined. Over-expression of95 in MCF7 induced serum deprivation cell death, as was assessed bymeasuring lactate dehydrogenase (LDH) activity released from cells, by aspectrophometric method (FIG. 3).

[0347] Conditioned Medium from MCF7-95 Inducible Clones Promoted CellDeath

[0348] In order to determine whether MCF7-95 conditioned medium canstimulate apoptosis in other cells, conditioned medium was collectedfrom MCF7-95 inducible clones that were grown in the presence or absenceof tetracycline, and was added to human umbilical vein endothelial cells(HUVEC). After incubation of 24 hours, HUVEC cell death was measured.Conditioned medium from MCF7 clones overexpressing 95 promoted HUVECcell death. The same phenomena was observed by adding the MCF7-95conditioned medium to non-transfected MCF7 and PC12 cells.

[0349] 95 Overexpression Protected MCF7 cells Against Hypoxia andH₂O₂-Induced Cell Death

[0350] To find the roles of 95 in hypoxia-induced cell death, theinducible clones were grown under ischemic conditions in the presence orabsence of tetracycline. 95 overexpression protected MCF7 cells againsthypoxia-induced cell death, as was assessed by measuring lactatedehydrogenase (LDH) activity released from cells (FIG. 4).

[0351] H₂O₂ is a natural product of metabolism, but at sufficientconcentrations it produces cell damage. To demonstrate whether H₂O₂induces apoptosis in MCF7-95 inducible clones, the cells were treatedwith 1 mM H₂O₂ for 24 hours. As shown in FIG. 5, 95 overexpressionprotected MCF7 cells against H₂O₂ induced apoptosis.

[0352] 95 Expression Was Up-regulated in the Brain of a Rat Model ofStroke

[0353] The ³⁵S-labeled probe specific to the gene 95 was hybridized tocoronal section of rat brains fixed at different time points (30minutes, 1 hour, 2 hours, 4 hours, 12 hours, 24 hours, 48 hours, 72hours) after permanent middle cerebral artery occlusion (MCAO). Resultsof this in situ hybridization study revealed the expression of the gene95 at 12 and 24 hours after MCAO. Hybridization signal located to thesubset of neurons in the transitional zone between the ischemic core andperi-infarct area.

[0354] 95 Expression Was Up-regulated in Tumors (Not Necessarily Human)

[0355] Sections of tumors grown from C6 glioma cells in nude mice werehybridized to ³⁵S-labeled riboprobe specific to the gene 95. Results ofin situ hybridization demonstrated expression of the 95 gene in tumorcells surrounding necrotic areas. This pattern of expression closelyresembles that of the VEGF revealed by hybridization of thecorresponding probe to the parallel sections. These results suggestactivation of the gene 95 expression in hypoxic areas within growingtumors.

[0356] Discussion

[0357] HIF-1 is a major regulator of the adaptation of the cells tohypoxic conditions. As observed with ES cells, this transcription factoris necessary to maintain cell proliferation in hypoxia (Iyer et al,1997). However, HIF-1 is also involved in apoptosis in ES cells(Carmeliet et al, 1998). In hypoxia, HIF-1α is stabilized and is able tointeract with p53. This interaction leads to stabilization and increasedcellular p53, which, however, inhibits HIF-1αactivity (Blagosklonny etal, 1998; An et al, 1998). Coexpression of HIF-1α and wild-type p53leads to the inhibition of the HIF-1-induced transactivation(Blagosklonny et al, 1998). The dual role of HIF-1 as a necessary factorin survival to hypoxic stresses, but also as a pro-apoptotic protein, isnot clear.

[0358] A novel p53 and HIF-1 target gene, 95 has been isolated andcharacterized. It shares homology with PA26, a member of the GADDfamily. 95 is up-regulated and induces DNA damaged apoptosis in a p53dependent manner. In contrast, 95 is up-regulated and protects MCF7cells against ischemia and H₂O₂-induced cell death.

[0359] Hydrogen peroxide (H₂O₂) has been known to activate themitochondrial permeability transition pore and the release of themitochondrial protein cytochrome c (Stridh et al, 1998; Sugano et al,1999). In the cytosol, cytochrome c in combination with Apaf-1 activatescaspase-9, which then finally leads to activation of caspase-3 andapoptosis (Hampton et al, 1997). Caspases are evolutionarily conservedexecutioners of programmed cell death in normal development and are alsoimplicated in a variety of pathological conditions, including cerebralischemia (Nicholson et al, 1997). A recent study provides in vitro andin vivo evidence that a family of caspases plays a pivotal role in thehypoxia- and ischemia-induced death of oligodendrocytes (Shibata et al,2000). The present results suggest that 95 is up-regulated by hypoxia,brain ischemia and H₂O₂, and that it plays a suppressive role inischemia- and H₂O₂-induced apoptosis. Further investigation will benecessary to determine whether caspases are involved in 95 apoptoticmachinery.

[0360] Bcl-2, a 26-kDa membrane-anchored proto-oncoprotein, was thefirst gene product discovered as an apoptosis suppressor acting invarious cells (Reed et al, 1994). After cerebral ischemia, Bcl-2 isinduced in surviving neurons (Clark et al, 1997), suggesting itsprotective effect on ischemic brain injury. Overexpression of Bcl-2 bygene transfer or in transgenic mice reduces the volume of infarctionafter cerebral ischemia (Martinou et al, 1994; Lawrence et al, 1996).Two mechanisms can be involved in the pro-survival effect of Bcl-2against ischemic insults. The first is the anti-apoptotic effect ofBcl-2 and the second is its function as an antioxidant (Hockenbery etal, 1993). Recently, it was shown that ischemic insults dephosphorylatedBcl-2 in a time-dependent fashion without affecting the total amount ofprotein, and suggested that dephosphorylation of serine 70 is one of thecritical factors in decreasing the anti-apoptotic function of Bcl-2(Itakura et al, 2000). The present results show that overexpression of95 in MCF7 cells induces dephosphorylated-Bcl-2 expression, and suggestthat dephosphorylation of Bcl-2 may be involved in 95-induced apoptosis.

[0361] Several previous studies have implicated GADD153 expression inthe mechanism of growth arrest and apoptosis (Barone et al, 1994; Chenet al, 1996). Introduction of GADD153 gene into gastric cancer cells canmodulate sensitivity to anticancer agents in association with apoptosis(Kim et al, 1999). Furthermore, loss of GADD 153 gene expression leadsto high genetic instability of oral melanoma cells (Korabiowska et al,1999). In this study, it was shown that introduction of 95 gene intohuman epithelial breast carcinoma MCF7 cells can modulate theirsensitivity to the anticancer agent doxorubicin. 95 has been mapped to1p34-35 (HTGS), a part of chromosome 1 frequently deleted in high stageneuroblastoma tumors and sporadic breast tumors (Jogi et al, 2000;Phelan et al, 1996). Future mutations analysis of 95 in neuroblastomaand breast tumor samples will answer whether 95 is likely to be involvedin the genesis of these tumors.

[0362] GADD153 and 95 could possess functions analogous to traditionalstress-response genes, serving to protect cells from stress-induceddamage and/or aiding the recovery of normal cellular functions followingstress. One way in which p53 is thought to potentiate genomic stability,and consequently inhibit tumorigenesis is the removal of damaged cellsthrough the triggering of apoptosis via transcriptional induction ofgenes that encode proapoptotic factors, such as 95. This study suggeststhat 95 induces DNA damage mediated apoptosis in a p53 dependent mannerand protects against oxidative stress mediated apoptosis in a p53independent mechanism. The identification of key events in the apoptoticpathway that are affected by cellular responses, such as the expressionof 95, could facilitate the identification of targets for themanipulation of this protein, which may have important medicalimplications.

[0363] Accordingly, it is clear that 95 is a good gene and has all ofthe utilities discussed herein for good genes. Promotion of apoptosis inDNA damaged cells is also a beneficial property. Thus, administration ofthe 95 gene product to the site of a hypoxic event will help toameliorate the undesirable effects of such an event.

EXAMPLE 3 98 SEQ ID NO: 5

[0364] Northern Blot Analysis

[0365] Expression of gene 98 is strongly up-regulated by hypoxia alreadyafter four hours of exposure. On Northern blots, it appears as a singlemRNA species of 4.4. Kb.

[0366] Cloning

[0367] A full-length 98 cDNA was cloned. It is 4138 bp long and containsan single ORF encompassing the nucleotides 204-1445. The putativeprotein is 414 amino acids long.

[0368] Bioinformatic Analysis

[0369] Search of the public databases revealed that 98 encoded proteinis similar to two other human proteins: (1) a putative protein encodedby anonymous human 24945 mRNA sequence (AF131826) and (2) VDUP1 (proteininduced in HL-60 cells by dihydroxy vitamin D3 treatment) (S73591). Nosignificant structural features were found by existing protein analysistools within the 98 putative protein.

[0370] It was previously demonstrated that treatment with vitamin D3 caninduce apoptosis in C6 rat glioma cells (Baudet et al, 1996). Therefore,the relationship between the vitamin D-induced cell killing and 98 geneexpression and function in glioma cells was studied.

[0371] The mammalian 98 expression vector was then prepared and itseffects studied.

EXAMPLE 4 60F6 SEQ ID NO: 6

[0372] Northern Blot Analysis

[0373] Expression of this gene is moderately up-regulated after 16 hoursof hypoxia. On Northern blot, it appears as a single 3.0 Kb species.

[0374] Cloning

[0375] A complete 60F6 human cDNA clone was isolated from A172 cDNAlibrary. The contig is 2675 bp long and contains a single ORF (bp134-866) able to code for a putative protein of 244 amino acids (SEQ IDNO: 7).

[0376] Bioinformatic Analysis

[0377] A similarity search against the public databases revealed thatthe N-terminal half of 60F6 sequence exactly corresponds to a human cDNAcoding for RhoE/Rho8 small GTP-binding protein (P52199, HSRHO8GRN). Theidentity of gene 60F6 was not determined before, because the smallsequenced fragment that was initially possessed, originated from theRho8 long 3′ UTR. All the sequence information available in publicdatabases did not include the long 3′ UTR of Rho8. Structurally, Rho8belongs to a family of Ras-related GTPases that regulate the actincytoskeleton. However, this protein is unique in that it isconstitutively active: GTPase deficient and in vivo farnesylated (Fosteret al, 1996). Therefore, it is intriguing to find that thisconstitutively active G-protein is regulated on the level oftranscription. Hypoxia regulation of Rho8 was not previously described.

EXAMPLE 5 648 Lysyl Hydroxylase 2SEQ ID NO: 8, 10 and 12

[0378] Northern analysis

[0379] Probe 648 has detected a single 3.8 Kb transcript on Northernblots. Expression was induced in C6 glioma cells already after 4 hoursof hypoxia.

[0380] Cloning

[0381] After extension of initial cDNA probe by RACE it became evidentthat identified rat sequence (SEQ ID NO: 8), encoding a 758 aa of SEQ IDNO: 9, is able to code for protein that represents a rat homologue ofhuman lysyl hydroxylase 2 (PLOD2). The full-length open reading frameswere cloned for both human (SEQ ID NO: 10) and rat (SEQ ID NO: 12) lysylhydroxylase 2 homologues (by PCR, using primers built on the basis ofknown sequence, for human variant, and degenerative primers, for ratvariant). The encoded proteins (SEQ ID NOs: 11 and 13, respectively)have well defined signal peptides.

[0382] Bioinformatics Data

[0383] The cloned rat 648 CDNA contains an ORF coding for a putativeprotein that is 88% identical to the published human PLOD2 sequences.The least conserved sequences are within the signal peptide, however itsfunctional features are completely preserved. The cloned human cDNA isalmost identical to published human PLOD2 sequence. The word “almost” inthe previous sentence stems from the fact that both in human and in ratcDNA species cloned in the inventors' laboratory a stretch of aminoacids between positions 501-521 of published sequence PLOD2 sequence wasabsent. Therefore, the present PLOD2 variants are differentiallyspliced. Both rat and human homologues were amplified from RNA extractedfrom glioma cell lines cultured in hypoxic conditions.

[0384] Literature Review

[0385] Lysyl hydroxylases are the enzymes that catalyze the formation ofhydroxylysine in collagens and other proteins with collagen-likeamino-acid sequences, by the hydroxylation of lysine residue in X-K-Gsequences. The hydroxylysine residues have two important functions: (1)serve as sites of attachment of carbohydrate units, and (2) they areessential for the stability of the intermolecular collagen crosslinks.Congenital deficiency of lysyl hydroxylase in humans leads to increasedsolubility of collagens and, consequently, to numerous defects inorganization of connective tissue in various organs. There are threeknown isoforms of lysyl hydroxylase, encoded by different genes. Inhumans, PLOD2 was found to be highly expressed in pancreas, skeletalmuscle, heart and placenta (by Northern blot). Nothing is known eitherabout the regulation of PLOD2 expression by hypoxia or about itsinvolvement in angiogenesis and tumorigenesis. Induction of PLOD2 byhypoxia can probably account for hypoxia-induced tissue fibrosis.Indeed, specific lysyl hydroxylase inhibitor, minoxidil, was able tosuppress both cellular collagen production and fibroblasts proliferation(Murad et al, 1987; Saika et al, 1995). There were suggestions inliterature to use modified lysyl hydroxylase inhibitor for treatment ofvitreoretinopathy (Handa et al, 1993).

[0386] Analysis of Alternatively Spliced Versions of Gene 648

[0387] In order to establish whether the observed alternative splicingof PLOD2 is regulated by hypoxia, a set of PCR primers were synthesizedthat flank the alternatively spliced region. The expected sizes ofRT-PCR products are: 216 bp, for published sequence and 156 bp, for thepresent sequence. Semi-quantitative RT-PCR was performed on RNA templateextracted from human glioma A172 cell culture in either normoxia or inhypoxia for 4 and 16 hours. The F obtained results clearly demonstratethat both PLOD2 forms are hypoxia regulated, but the form of theinvention appears only in hypoxic conditions.

[0388] Testing Potential Pro- and Antiapoptotic Activity in TransientTransfection Assays

[0389] pcDNA3-648 was transiently co-transfected together withpcDNA3-GFP in Hela and 293 cells. 24 and 48 hours later the cells werefixed and stained with DAPI. No apoptotic effect was observed in thetransfected cells. In order to evaluate the anti-apoptotic properties ofthe 648 protein, a co-transfection assay was conducted using thepcDNA3-GFP and the FAS plasmids. No anti-apoptotic effect was observed.

[0390] Obtaining Stable Cell Clones Overexpressing 648 cDNA

[0391] C6 were stably transfected with 5 μg of the pcDNA3-648 plasmid.Following G418 selection the level of expression was measured usingNorthern blot analysis in comparison to its level in C6 cells after 16hours under hypoxic conditions. Out of 18 independent clones from thepcDNA3-648 transfection, no one was positive.

[0392] In situ Hybridization Analysis

[0393] Retinopathy Model

[0394] Probe 648 demonstrates clear hybridization signal throughout theinner nuclear layer of “hypoxic” pup's retina while “normoxic” retina isnegative for the expression. No hybridization signal was detected inadult retina.

[0395] In mouse embryo sections hybridization signal was detected insome apoptotic cells in the roof of the fourth brain ventricle and indeveloping retina ganglia, where expressing cells had no apoptoticfeatures.

[0396] Multi-tissue block hybridization shows expression of 648 gene(rat PLOD2) in visceral smooth muscles in oviduct, uterus, stomach andintestine. Vascular smooth muscles do not display hybridization signal.

[0397] The most prominent cell type hybridizing to 648 probe in theovary are granulosa cells of larger secondary follicles. Nohybridization signal is detected in granulosa cells of primary and smallsecondary follicles. Significantly, hybridization signal is weakened inpostovulatory follicles and completely disappears in corpora lutea. Thisshows that expression in granulosa cells is established at later stagesof follicular maturation and it is abruptly down-regulated uponovulation and the onset of conversion into lutein cells. On the otherhand, follicular involution is not accompanied by the changes in 648expression since strong hybridization signal is preserved in granulosacells of atretic follicles.

[0398] Weak hybridization signal can be seen in some stromal cellssurrounding large secondary follicles and corpora lutea as well as incells of theca internal of secondary follicles. Prominent signal isfound in “interstitial glands”. This shows distinct regulation of 648expression in theca cells undergoing “luteinization” in differentlocations: it is down regulated in corpora lutea but preserved or evenup-regulated in interstitial glands.

[0399] As to the germinal cells, an oocyte that expresses 648 was foundonly in one primary follicle while many other primary and secondaryfollicles had no hybridization signal. This shows a transient expressionof 648 in oocytes at some stage of their development.

[0400] Discrepancy in the hybridization patterns of human (published)and rat PLOD2 (648) genes is explained by different sensitivities ofdifferent detection methods (Northern blot vs. in situ hybridization).The rat probe used in the present invention does not span analternatively spliced region.

EXAMPLE 6 24D4 SEQ ID NO: 14

[0401] Northern Blot Analysis

[0402] Expression of gene 24D4 is down-regulated after 16 hours ofhypoxia. On Northern blots, it appears as a single 1.5 Kb mRNA species.

[0403] Cloning

[0404] A partial 24D4 human cDNA clone was isolated from A172 CDNAlibrary. The available sequence is 1486 bp long and contains anN-terminal truncated ORF (bp 1-396), encoding the peptide of SEQ ID NO:15.

[0405] Bioinformatic Analysis

[0406] The sequence has no analogs in public databases. The availableprotein sequence contains three consequent Zn-finger motifs, all of C2H2type (aa 52-72, 80-100 and 108-128). Zinc finger domains of this typeare usually found in nucleic acid-binding proteins.

EXAMPLE 7 77H4 SEQ ID NO: 16

[0407] Northern Blot Analysis

[0408] Expression of gene 77H4 is up-regulated after 16 hours ofhypoxia. On Northern blots, it appears as a single mRNA species 0.6-0.7Kb in size.

[0409] Cloning

[0410] Several EST cDNA clones from public databases, corresponding toclone 77H4, were sequenced. All clones possess a poly A tail and apolyadenylation signal at their 3′ end.

[0411] Bioinformatic Analysis

[0412] Gene 77H4 (SEQ ID NO: 16) encodes a 360 bp protein (SEQ ID NO:17). An exhaustive search was performed of public databases for all77H4-related sequences. Several independent contigs were identified inTIGR THC database. All of them are not completely identical to oneanother and contain nucleotide deletions of various length. This shows acertain variability in 77H4 nucleotide sequence.

[0413] Recently, a novel steroid receptor transcriptional coactivator,SRA, was found to be present as an RNA molecule in the transcriptionactivating complex SRC-1 (Lanz et al, 1999). Although no similarity wasfound between clone 77H4 and SRA RNA on the sequence level, severalcharacteristic features seem to be shared by both sequences:

[0414] both mRNAs, 77H4 and SRA, are approximately of the same size—0.7Kb;

[0415] sequencing multiple cDNA clones corresponding to either mRNArevealed extensive variability in certain regions;

[0416] hybridization signals of both mRNA, therefore, appear as fuzzybands on Northern blots;

[0417] neither mRNA exhibit characteristics of protein.

[0418] Therefore, the 77H4 CDNA clone has similar to SRA function andcan serve a coactivator in some transcriptional complexes.

EXAMPLE 8 14G2 SEQ ID NO: 18

[0419] Northern Blot Analysis

[0420] Expression of gene 14G2 is regulated within 16 hours of hypoxia.On Northern blots, it appears as a single mRNA species.

[0421] Cloning

[0422] A partial 14G2 human CDNA clone was isolated. The availablesequence was then characterized and cloned as shown in SEQ ID NO: 18.

EXAMPLE 9 29F3 SEQ ID NO: 19

[0423] Northern Blot Analysis

[0424] Expression of gene 29F3 is regulated within 16 hours of hypoxia.On Northern blots, it appears as a single mRNA species.

[0425] Cloning

[0426] A partial 29F3 human cDNA clone was isolated. The availablesequence was then characterized and cloned as shown in SEQ ID NO: 19.

[0427] Throughout this application, various publications, includingUnited States patents, are referenced by author and year and patents bynumber. Full citations for the publications are listed below. Thedisclosures of these publications and patents in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.

[0428] The invention has been described in an illustrative manner, andit is to be understood that the terminology which has been used isintended to be in the nature of words of description rather than oflimitation.

[0429] Obviously, many modifications and variations of the presentinvention are possible in light of the above teachings. It is,therefore, to be understood that within the scope of the describedinvention, the invention can be practiced otherwise than as specificallydescribed.

[0430] The foregoing description of the specific embodiments will sofully reveal the general nature of the invention that others can, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without undue experimentation andwithout departing from the generic concept, and, therefore, suchadaptations and modifications should and are intended to be comprehendedwithin the meaning and range of equivalents of the disclosedembodiments. It is to be understood that the phraseology or terminologyemployed herein is for the purpose of description and not of limitation.The means, materials, and steps for carrying out various disclosedfunctions may take a variety of alternative forms without departing fromthe invention. Thus the expressions “means to . . . ” and “means for . .. ”, or any method step language, as may be found in the specificationabove and/or in the claims below, followed by a functional statement,are intended to define and cover whatever structural, physical, chemicalor electrical element or structure, or whatever method step, which maynow or in the future exist which carries out the recited function,whether or not precisely equivalent to the embodiment or embodimentsdisclosed in the specification above, i.e., other means or steps forcarrying out the same functions can be used; and it is intended thatsuch expressions be given their broadest interpretation.

[0431] The sequence list attached hereto is hereby incorporated byreference.

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[0627] P52199: Rho-Related GTP-Binding Protein RHOE (RHO8); LOCUSRHOE-HUMAN; submitted Dec. 15, 1998

[0628] S73591: MG068 homolog D02 _orf346-Mycoplasma pneumonia (strainATCC 29342); LOCUS: S73591; originally submitted Feb. 27, 1997; lastrevision Dec. 7, 1998

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 21 <210> SEQ ID NO 1<211> LENGTH: 1655 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (265)..(1575) <221>NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223> OTHER INFORMATION:“n” is unknown <221> NAME/KEY: misc_feature <222> LOCATION: (22)..(22)<223> OTHER INFORMATION: “n” is unknown <221> NAME/KEY: misc_feature<222> LOCATION: (41)..(41) <223> OTHER INFORMATION: “n” is unknown <221>NAME/KEY: misc_feature <222> LOCATION: (47)..(47) <223> OTHERINFORMATION: “n” is unknown <400> SEQUENCE: 1 gcacnaggtg tgtggcagcaanagccgcca gttcgggacc nccgcanctg gggtggcaac 60 ggcgcaggag gggtcgcggggagggagtgg tgagcgcagg cggcaggggt ctgggaaaga 120 cgaagtcgct atttgctgtctgagcgcgct cgcagctcct ggaagtgttg ccgcctctcg 180 gtttcgctct cgctcgctgcgctcctagaa ggggcggccg cctccaggac tgaccagggc 240 caagtggcgc tcggcgggcactac atg gcg gag ggt gaa ggg tac ttc gcc 291 Met Ala Glu Gly Glu Gly TyrPhe Ala 1 5 atg tct gag gac gag ctg gcc tgc agc ccc tac atc ccc cta ggcggc 339 Met Ser Glu Asp Glu Leu Ala Cys Ser Pro Tyr Ile Pro Leu Gly Gly10 15 20 25 gac ttc ggc ggc ggc gac ttc ggc ggc ggc gac ttc ggc ggt ggcggc 387 Asp Phe Gly Gly Gly Asp Phe Gly Gly Gly Asp Phe Gly Gly Gly Gly30 35 40 agc ttc ggt ggg cat tgc ttg gac tat tgc gaa agc cct acg gcg cac435 Ser Phe Gly Gly His Cys Leu Asp Tyr Cys Glu Ser Pro Thr Ala His 4550 55 tgc aat gtg ctg aac tgg gag caa gtg cag cgg ctg gac ggc atc ctg483 Cys Asn Val Leu Asn Trp Glu Gln Val Gln Arg Leu Asp Gly Ile Leu 6065 70 agc gag acc att ccg att cac ggg cgc ggc aac ttc ccc acg ctc gag531 Ser Glu Thr Ile Pro Ile His Gly Arg Gly Asn Phe Pro Thr Leu Glu 7580 85 ctg cag ccg agc ctg atc gtg aag gtg gtg cgg cgg cgc ctg gcc gag579 Leu Gln Pro Ser Leu Ile Val Lys Val Val Arg Arg Arg Leu Ala Glu 9095 100 105 aag cgc att ggc gtc cgc gac gtg cgc ctc aac ggc tcg gca gccagc 627 Lys Arg Ile Gly Val Arg Asp Val Arg Leu Asn Gly Ser Ala Ala Ser110 115 120 cat gtc ctg cac cag gac agc ggc ctg ggc tac aag gac ctg gacctc 675 His Val Leu His Gln Asp Ser Gly Leu Gly Tyr Lys Asp Leu Asp Leu125 130 135 atc ttc tgc gcc gac ctg cgc ggg gaa ggg gag ttt cag act gtgaag 723 Ile Phe Cys Ala Asp Leu Arg Gly Glu Gly Glu Phe Gln Thr Val Lys140 145 150 gac gtc gtg ctg gac tgc ctg ttg gac ttc tta ccc gag ggg gtgaac 771 Asp Val Val Leu Asp Cys Leu Leu Asp Phe Leu Pro Glu Gly Val Asn155 160 165 aaa gag aag atc aca cca ctc acg ctc aag gaa gct tat gtg cagaaa 819 Lys Glu Lys Ile Thr Pro Leu Thr Leu Lys Glu Ala Tyr Val Gln Lys170 175 180 185 atg gtt aaa gtg tgc aat gac tct gac cga tgg agt ctt atatcc ctg 867 Met Val Lys Val Cys Asn Asp Ser Asp Arg Trp Ser Leu Ile SerLeu 190 195 200 tca aac aac agt ggc aaa aat gtg gaa ctg aaa ttt gtg gattcc ctc 915 Ser Asn Asn Ser Gly Lys Asn Val Glu Leu Lys Phe Val Asp SerLeu 205 210 215 cgg agg cag ttt gaa ttc agt gta gat tct ttt caa atc aaatta gac 963 Arg Arg Gln Phe Glu Phe Ser Val Asp Ser Phe Gln Ile Lys LeuAsp 220 225 230 tct ctt ctg ctc ttt tat gaa tgt tca gag aac cca atg actgag aca 1011 Ser Leu Leu Leu Phe Tyr Glu Cys Ser Glu Asn Pro Met Thr GluThr 235 240 245 ttt cac ccc aca ata atc ggg gag agc gtc tat ggc gat ttccag gaa 1059 Phe His Pro Thr Ile Ile Gly Glu Ser Val Tyr Gly Asp Phe GlnGlu 250 255 260 265 gcc ttt gat cac ctt tgt aac aag atc att gcc acc aggaac cca gag 1107 Ala Phe Asp His Leu Cys Asn Lys Ile Ile Ala Thr Arg AsnPro Glu 270 275 280 gaa atc cga ggg gga ggc ctg ctt aag tac tgc aac ctcttg gtg agg 1155 Glu Ile Arg Gly Gly Gly Leu Leu Lys Tyr Cys Asn Leu LeuVal Arg 285 290 295 ggc ttt agg ccc gcc tct gat gaa atc aag acc ctt caaagg tat atg 1203 Gly Phe Arg Pro Ala Ser Asp Glu Ile Lys Thr Leu Gln ArgTyr Met 300 305 310 tgt tcc agg ttt ttc atc gac ttc tca gac att gga gagcag cag aga 1251 Cys Ser Arg Phe Phe Ile Asp Phe Ser Asp Ile Gly Glu GlnGln Arg 315 320 325 aaa ctg gag tcc tat ttg cag aac ctc ttt gtg gga ttggaa gcc cgc 1299 Lys Leu Glu Ser Tyr Leu Gln Asn Leu Phe Val Gly Leu GluAla Arg 330 335 340 345 aag tat gag tat ctc atg acc ctt cat gga gtg gtaaat gag agc tca 1347 Lys Tyr Glu Tyr Leu Met Thr Leu His Gly Val Val AsnGlu Ser Ser 350 355 360 gtg tgc ctg atg gga cat gaa aga aga cag act ttaaac ctt atc acc 1395 Val Cys Leu Met Gly His Glu Arg Arg Gln Thr Leu AsnLeu Ile Thr 365 370 375 atg ctg gct atc cgg gtg tta gct gac caa aat gtcatt cct aat gtg 1443 Met Leu Ala Ile Arg Val Leu Ala Asp Gln Asn Val IlePro Asn Val 380 385 390 gct aat gtc act tgc tat tac cag cca gcc ccc tatgta gca gat gcc 1491 Ala Asn Val Thr Cys Tyr Tyr Gln Pro Ala Pro Tyr ValAla Asp Ala 395 400 405 aac ttt agc aat tac tac att gca cag gtt cag ccagta ttc acg tgc 1539 Asn Phe Ser Asn Tyr Tyr Ile Ala Gln Val Gln Pro ValPhe Thr Cys 410 415 420 425 cag caa cag acc tac tcc act tgg cta ccc tgcaat taagaatcat 1585 Gln Gln Gln Thr Tyr Ser Thr Trp Leu Pro Cys Asn 430435 ttaaaaatgt cctgtgggga agccatttca gacaagacag gagagaaaaa aaaaaaaaaa1645 aaaaaaaaaa 1655 <210> SEQ ID NO 2 <211> LENGTH: 437 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met Ala Glu Gly Glu GlyTyr Phe Ala Met Ser Glu Asp Glu Leu Ala 1 5 10 15 Cys Ser Pro Tyr IlePro Leu Gly Gly Asp Phe Gly Gly Gly Asp Phe 20 25 30 Gly Gly Gly Asp PheGly Gly Gly Gly Ser Phe Gly Gly His Cys Leu 35 40 45 Asp Tyr Cys Glu SerPro Thr Ala His Cys Asn Val Leu Asn Trp Glu 50 55 60 Gln Val Gln Arg LeuAsp Gly Ile Leu Ser Glu Thr Ile Pro Ile His 65 70 75 80 Gly Arg Gly AsnPhe Pro Thr Leu Glu Leu Gln Pro Ser Leu Ile Val 85 90 95 Lys Val Val ArgArg Arg Leu Ala Glu Lys Arg Ile Gly Val Arg Asp 100 105 110 Val Arg LeuAsn Gly Ser Ala Ala Ser His Val Leu His Gln Asp Ser 115 120 125 Gly LeuGly Tyr Lys Asp Leu Asp Leu Ile Phe Cys Ala Asp Leu Arg 130 135 140 GlyGlu Gly Glu Phe Gln Thr Val Lys Asp Val Val Leu Asp Cys Leu 145 150 155160 Leu Asp Phe Leu Pro Glu Gly Val Asn Lys Glu Lys Ile Thr Pro Leu 165170 175 Thr Leu Lys Glu Ala Tyr Val Gln Lys Met Val Lys Val Cys Asn Asp180 185 190 Ser Asp Arg Trp Ser Leu Ile Ser Leu Ser Asn Asn Ser Gly LysAsn 195 200 205 Val Glu Leu Lys Phe Val Asp Ser Leu Arg Arg Gln Phe GluPhe Ser 210 215 220 Val Asp Ser Phe Gln Ile Lys Leu Asp Ser Leu Leu LeuPhe Tyr Glu 225 230 235 240 Cys Ser Glu Asn Pro Met Thr Glu Thr Phe HisPro Thr Ile Ile Gly 245 250 255 Glu Ser Val Tyr Gly Asp Phe Gln Glu AlaPhe Asp His Leu Cys Asn 260 265 270 Lys Ile Ile Ala Thr Arg Asn Pro GluGlu Ile Arg Gly Gly Gly Leu 275 280 285 Leu Lys Tyr Cys Asn Leu Leu ValArg Gly Phe Arg Pro Ala Ser Asp 290 295 300 Glu Ile Lys Thr Leu Gln ArgTyr Met Cys Ser Arg Phe Phe Ile Asp 305 310 315 320 Phe Ser Asp Ile GlyGlu Gln Gln Arg Lys Leu Glu Ser Tyr Leu Gln 325 330 335 Asn Leu Phe ValGly Leu Glu Ala Arg Lys Tyr Glu Tyr Leu Met Thr 340 345 350 Leu His GlyVal Val Asn Glu Ser Ser Val Cys Leu Met Gly His Glu 355 360 365 Arg ArgGln Thr Leu Asn Leu Ile Thr Met Leu Ala Ile Arg Val Leu 370 375 380 AlaAsp Gln Asn Val Ile Pro Asn Val Ala Asn Val Thr Cys Tyr Tyr 385 390 395400 Gln Pro Ala Pro Tyr Val Ala Asp Ala Asn Phe Ser Asn Tyr Tyr Ile 405410 415 Ala Gln Val Gln Pro Val Phe Thr Cys Gln Gln Gln Thr Tyr Ser Thr420 425 430 Trp Leu Pro Cys Asn 435 <210> SEQ ID NO 3 <211> LENGTH: 3454<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (323)..(1762) <221> NAME/KEY: misc_feature<222> LOCATION: (606)..(606) <223> OTHER INFORMATION: “n” is unknown<221> NAME/KEY: misc_feature <222> LOCATION: (2561)..(2561) <223> OTHERINFORMATION: “n” is unknown <221> NAME/KEY: misc_feature <222> LOCATION:(2594)..(2504) <223> OTHER INFORMATION: “n” is unknown <221> NAME/KEY:misc_feature <222> LOCATION: (2613)..(2613) <223> OTHER INFORMATION: “n”is unknown <400> SEQUENCE: 3 ctccgcggcg gggatgctga ggagcgctgg gtccgggagcagccctggcc cctgcggact 60 tccgaggccg tgaaaacccc tgcgctgcgg cccttcccaggcccccgagg ccgttcgccg 120 ttcccgaagc ccgactgggg gaagagtcca gcaccaaagcggccgttctc ggattccgga 180 gcgttctgga gccccgagag acgccccggg gttctagaagctccccggcg gcgcccagtc 240 ccggcttcat tcgggcgtcc ctccgaaacc cactcgggtgcacgggtcgt cggcgagccg 300 cgaccgggtc ctggcgcgca cc atg atc gtg gcg gactcc gag tgc cgc gca 352 Met Ile Val Ala Asp Ser Glu Cys Arg Ala 1 5 10gag ctc aag gac tac ctg cgg ttc gcc ccg ggc ggc gtc ggc gac tcg 400 GluLeu Lys Asp Tyr Leu Arg Phe Ala Pro Gly Gly Val Gly Asp Ser 15 20 25 ggcccc gga gag gag cag agg gag agc cgg gct cgg cga ggc cct cga 448 Gly ProGly Glu Glu Gln Arg Glu Ser Arg Ala Arg Arg Gly Pro Arg 30 35 40 ggg cccagc gcc ttc atc ccc gtg gag gag gtc ctt cgg gag ggg gct 496 Gly Pro SerAla Phe Ile Pro Val Glu Glu Val Leu Arg Glu Gly Ala 45 50 55 gag agc ctcgag cag cac ctg ggg ctg gag gca ctg atg tcc tct ggg 544 Glu Ser Leu GluGln His Leu Gly Leu Glu Ala Leu Met Ser Ser Gly 60 65 70 cga gta gac aacctg gca gtg gtg atg ggc ctg cac cct gac tac ttt 592 Arg Val Asp Asn LeuAla Val Val Met Gly Leu His Pro Asp Tyr Phe 75 80 85 90 acc agc ttc tggcnc ctg cac tac ctg ctg ctg cac acg gat ggt ccc 640 Thr Ser Phe Trp XaaLeu His Tyr Leu Leu Leu His Thr Asp Gly Pro 95 100 105 ttg gcc agc tcctgg cgc cac tac att gcc atc atg gct gcc gcc cgc 688 Leu Ala Ser Ser TrpArg His Tyr Ile Ala Ile Met Ala Ala Ala Arg 110 115 120 cat cag tgt tcttac ctg gta ggc tcc cac atg gcc gag ttt ctg cag 736 His Gln Cys Ser TyrLeu Val Gly Ser His Met Ala Glu Phe Leu Gln 125 130 135 act ggt ggt gaccct gag tgg ctg ctg ggc ctc cac cgg gcc ccc gag 784 Thr Gly Gly Asp ProGlu Trp Leu Leu Gly Leu His Arg Ala Pro Glu 140 145 150 aag ctg cgc aaactc agc gag atc aac aag ttg ctg gcg cat cgg cca 832 Lys Leu Arg Lys LeuSer Glu Ile Asn Lys Leu Leu Ala His Arg Pro 155 160 165 170 tgg ctc atcacc aag gaa cac atc cag gcc ttg ctg aag acc ggc gag 880 Trp Leu Ile ThrLys Glu His Ile Gln Ala Leu Leu Lys Thr Gly Glu 175 180 185 cac act tggtcc ctg gcc gag ctc att cag gct ctg gtc ctg ctc acc 928 His Thr Trp SerLeu Ala Glu Leu Ile Gln Ala Leu Val Leu Leu Thr 190 195 200 cac tgc cactcg ctc tcc tcc ttc gtg ttt ggc tgt ggc atc ctc cct 976 His Cys His SerLeu Ser Ser Phe Val Phe Gly Cys Gly Ile Leu Pro 205 210 215 gag ggg gatgca gat ggc agc cct gcc ccc cag gca cct aca ccc cct 1024 Glu Gly Asp AlaAsp Gly Ser Pro Ala Pro Gln Ala Pro Thr Pro Pro 220 225 230 agt gaa cagagc agc ccc cca agc agg gac ccg ttg aac aac tct ggg 1072 Ser Glu Gln SerSer Pro Pro Ser Arg Asp Pro Leu Asn Asn Ser Gly 235 240 245 250 ggc tttgag tct gcc cgc gac gtg gag gcg ctg atg gag cgc atg cag 1120 Gly Phe GluSer Ala Arg Asp Val Glu Ala Leu Met Glu Arg Met Gln 255 260 265 cag ctgcag gag agc ctg ctg cgg gat gag ggg acg tcc cag gag gag 1168 Gln Leu GlnGlu Ser Leu Leu Arg Asp Glu Gly Thr Ser Gln Glu Glu 270 275 280 atg gagagc cgc ttt gag ctg gag aag tca gag agc ctg ctg gtg acc 1216 Met Glu SerArg Phe Glu Leu Glu Lys Ser Glu Ser Leu Leu Val Thr 285 290 295 ccc tcagct gac atc ctg gag ccc tct cca cac cca gac atg ctg tgc 1264 Pro Ser AlaAsp Ile Leu Glu Pro Ser Pro His Pro Asp Met Leu Cys 300 305 310 ttt gtggaa gac cct act ttc gga tat gag gac ttc act cgg aga ggg 1312 Phe Val GluAsp Pro Thr Phe Gly Tyr Glu Asp Phe Thr Arg Arg Gly 315 320 325 330 gctcag gca ccc cct acc ttc cgg gcc cag gat tat acc tgg gaa gac 1360 Ala GlnAla Pro Pro Thr Phe Arg Ala Gln Asp Tyr Thr Trp Glu Asp 335 340 345 catggc tac tcg ctg atc cag cgg ctt tac cct gag ggt ggg cag ctg 1408 His GlyTyr Ser Leu Ile Gln Arg Leu Tyr Pro Glu Gly Gly Gln Leu 350 355 360 ctggat gag aag ttc cag gca gcc tat agc ctc acc tac aat acc atc 1456 Leu AspGlu Lys Phe Gln Ala Ala Tyr Ser Leu Thr Tyr Asn Thr Ile 365 370 375 gccatg cac agt ggt gtg gac acc tcc gtg ctc cgc agg gcc atc tgg 1504 Ala MetHis Ser Gly Val Asp Thr Ser Val Leu Arg Arg Ala Ile Trp 380 385 390 aactat atc cac tgc gtc ttt ggc atc aga tat gat gac tat gat tat 1552 Asn TyrIle His Cys Val Phe Gly Ile Arg Tyr Asp Asp Tyr Asp Tyr 395 400 405 410ggg gag gtg aac cag ctc ctg gag cgg aac ctc aag gtc tat atc aag 1600 GlyGlu Val Asn Gln Leu Leu Glu Arg Asn Leu Lys Val Tyr Ile Lys 415 420 425aca gtg gcc tgc tac cca gag aag acc acc cga aga atg tac aac ctc 1648 ThrVal Ala Cys Tyr Pro Glu Lys Thr Thr Arg Arg Met Tyr Asn Leu 430 435 440ttc tgg agg cac ttc cgc cac tca gag aag gtc cac gtg aac ttg ctg 1696 PheTrp Arg His Phe Arg His Ser Glu Lys Val His Val Asn Leu Leu 445 450 455ctc ctg gag gcg cgc atg caa gcc gct ctg ctg tac gcc ctc cgt gcc 1744 LeuLeu Glu Ala Arg Met Gln Ala Ala Leu Leu Tyr Ala Leu Arg Ala 460 465 470atc acc cgc tac atg acc tgactcctga gcaggacctg ggcccggttc 1792 Ile ThrArg Tyr Met Thr 475 480 agctccccac aaggacttct ctgtctggag acagccccagacccttttgt gtcccatgcc 1852 caccctcccc acgctgcagt gggcttgtgt gtgatgtgcagtcccgaagc cacaccctcc 1912 cttttcctca ctggaatgga cagttcattg cactgactctgggatctcag ccctgctcct 1972 gggagctgga agagcacttg gagatcctaa gggaccacacccttcctcct tcccctgccc 2032 acagaggcag agggcacagg aaagaagccg ggccaagctcggaattaatg tgccacaagt 2092 gttgtggcct tcctgaactg ggaagtccct ggctggcccccgggggagag gggcaaatgc 2152 ctccgggact gacactccag gcagctttgc cttctctcccctgtcatttc cagatttcat 2212 tacctcctac ttgccattca cccatcaatg tgaaagtcagggtcacagct ggtctgtgtg 2272 tccagttccc taaaagcctg ttctgttggg cagcctgaggctgttgcccg aatcctagtt 2332 cagttttttg acttcctttg ccctttttcc cttttctccatgcttaatgg tgtgaggcgt 2392 caggagagag gccaagtaca taaaaaaaaa aaaaagcagattatctctag agagtttgag 2452 cctttgctgg tcacattgcc ttctgaagag gagggagtattagattataa atcctcttta 2512 ttttggtcct ttatgcttga ggttccaacc tggagccacagtgtgtgana ggaggaggag 2572 agggagaatt ctgttctccc anagctgcac ctgcctcgcanaggccagca ccccactctc 2632 ctgcctccag tggccctgcc gcagatgtct cccaaaaagttgagcctttc tagatggctt 2692 aggtggcacc atggctcagc aggaggggcg ggaggcaccagggttcttgt ttggaccctg 2752 cccctgggcc atggccaggt gaccatggct acattgccaaacctctgact gccacagctg 2812 cagactgaga gggtgggtct gagtccccac aatgtctgaagctgcccctg ggattctcag 2872 gccaacctgc caacagcaag cggattttct tgcaagatcagggaccccat ttctgcagcc 2932 agtgtctcct gggtgccttc tgaggactcc cacccccatcccagtatctc atctgtcccc 2992 tctcctgggg cttaagtggg ttgcttccag gcagaagcagccaaggaccg attccaggca 3052 ctttctgtag caaatgactg tgaattacga cttctcttgcccttcttcta gcagtctgtg 3112 cctcctctct gaccagtttg gagggcactg aagaaaggcaagggccgtgc tgctgctggg 3172 cggggcagga gaggagcctg gccagtgtgc cacattaaatacccgtgcag gcgcggagaa 3232 gcaaccggca cccccttccg gcctgaaagc cctccctgcaagaaggtgtg caggagagaa 3292 gaggccccgg catggggatc tgggttctag agggcatgtgatgactgtaa atgttcactg 3352 ggtgggtagg gagtggtatc cagtgttcaa gtgcagaaatctttggcttt gctaccagtt 3412 ccatatgatg agaaataaac gttcgctgag gttttgtttcat 3454 <210> SEQ ID NO 4 <211> LENGTH: 480 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (118)..(118) <223> OTHER INFORMATION: “Xaa” is unknown <400>SEQUENCE: 4 Met Ile Val Ala Asp Ser Glu Cys Arg Ala Glu Leu Lys Asp TyrLeu 1 5 10 15 Arg Phe Ala Pro Gly Gly Val Gly Asp Ser Gly Pro Gly GluGlu Gln 20 25 30 Arg Glu Ser Arg Ala Arg Arg Gly Pro Arg Gly Pro Ser AlaPhe Ile 35 40 45 Pro Val Glu Glu Val Leu Arg Glu Gly Ala Glu Ser Leu GluGln His 50 55 60 Leu Gly Leu Glu Ala Leu Met Ser Ser Gly Arg Val Asp AsnLeu Ala 65 70 75 80 Val Val Met Gly Leu His Pro Asp Tyr Phe Thr Ser PheTrp Xaa Leu 85 90 95 His Tyr Leu Leu Leu His Thr Asp Gly Pro Leu Ala SerSer Trp Arg 100 105 110 His Tyr Ile Ala Ile Met Ala Ala Ala Arg His GlnCys Ser Tyr Leu 115 120 125 Val Gly Ser His Met Ala Glu Phe Leu Gln ThrGly Gly Asp Pro Glu 130 135 140 Trp Leu Leu Gly Leu His Arg Ala Pro GluLys Leu Arg Lys Leu Ser 145 150 155 160 Glu Ile Asn Lys Leu Leu Ala HisArg Pro Trp Leu Ile Thr Lys Glu 165 170 175 His Ile Gln Ala Leu Leu LysThr Gly Glu His Thr Trp Ser Leu Ala 180 185 190 Glu Leu Ile Gln Ala LeuVal Leu Leu Thr His Cys His Ser Leu Ser 195 200 205 Ser Phe Val Phe GlyCys Gly Ile Leu Pro Glu Gly Asp Ala Asp Gly 210 215 220 Ser Pro Ala ProGln Ala Pro Thr Pro Pro Ser Glu Gln Ser Ser Pro 225 230 235 240 Pro SerArg Asp Pro Leu Asn Asn Ser Gly Gly Phe Glu Ser Ala Arg 245 250 255 AspVal Glu Ala Leu Met Glu Arg Met Gln Gln Leu Gln Glu Ser Leu 260 265 270Leu Arg Asp Glu Gly Thr Ser Gln Glu Glu Met Glu Ser Arg Phe Glu 275 280285 Leu Glu Lys Ser Glu Ser Leu Leu Val Thr Pro Ser Ala Asp Ile Leu 290295 300 Glu Pro Ser Pro His Pro Asp Met Leu Cys Phe Val Glu Asp Pro Thr305 310 315 320 Phe Gly Tyr Glu Asp Phe Thr Arg Arg Gly Ala Gln Ala ProPro Thr 325 330 335 Phe Arg Ala Gln Asp Tyr Thr Trp Glu Asp His Gly TyrSer Leu Ile 340 345 350 Gln Arg Leu Tyr Pro Glu Gly Gly Gln Leu Leu AspGlu Lys Phe Gln 355 360 365 Ala Ala Tyr Ser Leu Thr Tyr Asn Thr Ile AlaMet His Ser Gly Val 370 375 380 Asp Thr Ser Val Leu Arg Arg Ala Ile TrpAsn Tyr Ile His Cys Val 385 390 395 400 Phe Gly Ile Arg Tyr Asp Asp TyrAsp Tyr Gly Glu Val Asn Gln Leu 405 410 415 Leu Glu Arg Asn Leu Lys ValTyr Ile Lys Thr Val Ala Cys Tyr Pro 420 425 430 Glu Lys Thr Thr Arg ArgMet Tyr Asn Leu Phe Trp Arg His Phe Arg 435 440 445 His Ser Glu Lys ValHis Val Asn Leu Leu Leu Leu Glu Ala Arg Met 450 455 460 Gln Ala Ala LeuLeu Tyr Ala Leu Arg Ala Ile Thr Arg Tyr Met Thr 465 470 475 480 <210>SEQ ID NO 5 <211> LENGTH: 4138 <212> TYPE: DNA <213> ORGANISM: Homosapiens <400> SEQUENCE: 5 gacgagcggg agcgcggagc agcagcctct gctgccctgactttttaaga aatctcaatg 60 aactatttgt agagaatcac tgatccggcc tgcaagcattttgcacggca aaaatatcga 120 tcagtgttaa gtgaagatca cattttatat gcgatcttgacttttttgtc ttacattata 180 tttttataga ttttgttata aacatggtgc tgggaaaggtgaagagtttg acaataagct 240 ttgactgtct taatgacagc aatgtccctg tgtattctagtggggatacc gtctcaggaa 300 gggtaaattt agaagttact ggggaaatca gagtaaaatctcttaaaatt catgcaagag 360 gacatgcgaa agtacgctgg actgaatcta gaaacgccggctccaatact gcctatacac 420 agaattacac tgaagaagta gagtatttca accataaagacatcttaatt gggcacgaaa 480 gagatgatga taattccgaa gaaggcttcc acactattcattcaggaagg catgaatatg 540 cattcagctt cgagcttcca cagacaccac tcgctacctcattcgaaggc cgacatggca 600 gtgtgcgcta ttgggtgaaa gccgaattgc acaggccttggctactacca gtaaaattaa 660 agaaggaatt tacagtcttt gagcatatag atatcaacactccttcatta ctgtcacccc 720 aagcaggcac aaaagaaaag acactctgtt gctggttctgtacctcaggc ccaatatcct 780 taagtgccaa aattgaaagg aagggctata ccccaggtgaatcaattcag atatttgctg 840 agattgagaa ctgctcttcc cgaatggtgg tgccaaaggcagccatttac caaacacagg 900 ccttctatgc caaagggaaa atgaaggaag taaaacagcttgtggctaac ttgcgtgggg 960 aatccttatc atctggaaag acagagacgt ggaatggcaagttgctgaaa attccaccag 1020 tttctccctc tatcctcgac tgtagtataa tccgcgtggaatattcacta atggtatatg 1080 tggatattcc tggagctatg gatttatttc ttaatttgccacttgtcatc ggtaccattc 1140 ctctacatcc atttggtagc agaacctcaa gtgtaagcagtcagtgtagc atgaatatga 1200 actggctcag tttatcactt cctgaaagac ctgaagcaccacccagctat gcagaagtgg 1260 taacagagga acaaaggcgg aacaatcttg caccagtgagtgcttgtgat gactttgaga 1320 gagcccttca aggaccactg tttgcatata tccaggagtttcgattcttg cctccacctc 1380 tttattcaga gattgatcca aatcctgatc agtcagcagatgatagacca tcctgcccct 1440 ctcgttgaag gaacacttgg ttgaatcaag ttgatgtgggttccgaactg tatctcttcc 1500 ggctgaggac agagaagtat cttggagaca cgtttcagaggaagtggaat tacttttgcc 1560 cagaaaaatg gcgaatacat gaaacaacca gtgatcatgctttagaagcc tacagcaaca 1620 ttctgagact gctccaacat gcttgaagat ctaagcttttctcttttaaa actggcacat 1680 actcagagca gtcttcttag cctatggtcg tacgtgtcaagacatcacgt tgtaaagagg 1740 gatgatttcc ttcttttgat ttgaaaattt gcacatgctcaatgcttaca ttgtgcggtt 1800 cgacgtcact acagcttctt tttttttttt tttttttctatttttgccag actcttgata 1860 ctcttaaaac ttgtttgtgg tcagcacaac aaggaacaaaacaaagcttt gaaaaaactt 1920 taacatgaaa aaacgcactg acattttttt ttatttaatatagcctggac tttacctgcg 1980 tatgcacatg ctcagaattg tctactaggc tgactatgtatcacctcttc agcttggatc 2040 caattgtgga tttatttaca aacatcaaat gccttcaagccaatcctttt tgctgtatgt 2100 tttgcagcct actgtagtag atacgcaaca gataatgtgggaaaaaaaga gataagagga 2160 ggaagctaat aagagactgt caagattgta taccttcttggtttctttta agaatttgtt 2220 gcctttctac tattacagca aagcagcatt ttgttactgactgcctaaaa tcacttaatc 2280 tcaggtgaac gcatcacttg ccaaactgtt ggaatgctatttgtgttttg ttgcactgtt 2340 tttttcgttt gtttgtttgt ttatttggtt ggctttttggagagggaaat ttggaaacgg 2400 gacatacaca aaagttacac acccacattc cctttttatcatgacataca agaagaaact 2460 agcagagcta agaatggagt gaagaaaggc agtatggcaggcaccagcaa agagttgagg 2520 gctgttgctc ttaaaaatta ttttttttat tattattttgaaagtatgga agttttccat 2580 tcactgggga aaggagggaa aagtgcattt atttttatacagagttactt aattacctcc 2640 aaaacacata tgttggaaat cgcttttgct ggtgcaaagtatattaatga gcaggaatac 2700 atacattgag gttatgaata gagagctcaa tttgtacctttgctgtcttg ctcaagcttg 2760 gtatggcatg aaaactcgac tttattccaa aagtaacttcaaaatttaaa atactagaac 2820 gtttgctgcg ataaatcttt tggatttttg tgtttttctaatgagaatac tgtttttcat 2880 tacctaaaga acaatttgct aaacatgaga aatcactcactttgattatg tatagattac 2940 ataggaagaa caatcacatc agtaagttat agtttatattaaaggtaatt ttctgttggc 3000 tcataacaaa tataccagca ttcatgatag catttcagcattttccaagg taccaagtgt 3060 acttattttg ttgttgttgt tgttgttgta ttttagaaggaattcagctc tgatgttttt 3120 aaagaaaacc agcatctctg atgttgcaac atacgtgtaaaatgggtgtt acatctatcc 3180 tgccatttaa ccccacagtt aataaagtgg ctgaaaataatagtagctct ggcttggtgc 3240 ttgacctggt taaatactgt cttaaagctc atacaaaacaaataggcttt tccataagtg 3300 gcctttaaga aaacatggaa gacaattcat gtttgacaaatgctgacagg gtgaagaaag 3360 cccagtgtaa aaatgaatcg cgttttaagt gattcggktaaagagtttgg gctcccgtag 3420 caaactaata ctagataata aggaaatggg ggtgaaatatttttttattg ttgaatcatt 3480 ttgtgaatgt ccccctcaaa aaaagctaat ggaatatttggcataaaggg catttggtgg 3540 ttttattttt gtttgagggg gattgtcaga aaatcccttttctctcttac gtctaactga 3600 ctagggaaca attgttgata tgcatagcat tggaatacttgtcattatat actcttacaa 3660 ataacacatg aagcaagaat gaccaatatt ctgataattggcactggatc acaaaatgtg 3720 ataaaacttt aaatgtataa aactttatca aataaagttttattttcccc tttaaaatgt 3780 atttctttag aggcattact tttttaaaaa tattggtcaattcctgacat aagatgtgag 3840 gttcacagtt gtattccagt attcaagata gattcctgatttttcaatta ggaaaagtaa 3900 aatccaaaat gttagcaaaa caaagtgcaa tattaaatgtttgctttata gattatattc 3960 tatggctgtt tgtaatttct ctttttttcc ttttttatttggtgctgaat atgtccttgt 4020 aggctctgtt ttaagaaaac aatatgtggg aaatgatttaatttttccta ttgctcttcc 4080 ttgtggaaaa taaagtgttt tgtttttttc tgttttgtaaaaaaaaaaaa aaaaaaaa 4138 <210> SEQ ID NO 6 <211> LENGTH: 1303 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (52)..(52) <223> OTHER INFORMATION: “n” isunknown <221> NAME/KEY: misc_feature <222> LOCATION: (92)..(92) <223>OTHER INFORMATION: “n” is unknown <221> NAME/KEY: misc_feature <222>LOCATION: (1212)..(1212) <223> OTHER INFORMATION: “n” is unknown <221>NAME/KEY: misc_feature <222> LOCATION: (1288)..(1288) <223> OTHERINFORMATION: “n” is unknown <221> NAME/KEY: misc_feature <222> LOCATION:(1290)..(1290) <223> OTHER INFORMATION: “n” is unknown <221> NAME/KEY:misc_feature <222> LOCATION: (1291)..(1291) <223> OTHER INFORMATION: “n”is unknown <221> NAME/KEY: misc_feature <222> LOCATION: (1293)..(1293)<223> OTHER INFORMATION: “n” is unknown <221> NAME/KEY: misc_feature<222> LOCATION: (1294)..(1294) <223> OTHER INFORMATION: “n” is unknown<221> NAME/KEY: misc_feature <222> LOCATION: (1295)..(1295) <223> OTHERINFORMATION: “n” is unknown <221> NAME/KEY: misc_feature <222> LOCATION:(1296)..(1296) <223> OTHER INFORMATION: “n” is unknown <400> SEQUENCE: 6cctcctcccc tcctcccgcc cacgcccctg ctccccgccc ccggaagccc cngcggaacg 60gttacgccgc gacgaagtaa gggtgggttc tnaaggaaag ccctttgcca atcttgcaag 120atttgtagac cagcactaca aagatcgcat agatcaaata ggaaaaaaaa tgtcgatttt 180tattcagtct gatggttctg ttcttcattg tgattgtcat taaaaagtgg taaattgctc 240aatgtaatat ttttgtgcgc tgtttagaag ttgtgtgatt ttttgccatc gttgataaaa 300atgcaaagtc aaataaaagg tgtcttggtt tgatgtcata gaatgatcca aggagagaaa 360aaaggtagtt actgttttca ccagaaaagg taatgagtga aggaaagaat agtagcagaa 420agcacagttt gtgagtaaag ctgtctggaa ttaagttacc aaaaatacaa agcaaaagga 480ctattatttt gggttgaagc tccaaaactg acagcatctg ataatctgtt ggtttatttc 540acttttcatt aaatgaacat tgatgagaga agatgccact tacccaagct ttagagaatc 600cctagtggaa gattatatga taaactttca gtcctgacat aacactaggg catttctaga 660gtgtcattgc taaaacctca ctgaacagac gcagccaagg tctgtgttca gcacttggtc 720tctgttgtta cgtaaaataa taagcattta aaatagttta cagatatttt tgaccagttc 780cttttagaga ttctttcaga gaagaaacca gatctgacct gtttattgtt ggcgcttgtt 840gaaaacgagc tttctttccc atgatagtgc ttcgtttttg aagtgttgaa gctgtgctcc 900ccttaaatcg tggcaggaga gattaaggta attacaacac tcagttctat gtcttacaag 960cactttgtct tgtctctgca agaaaattcg attccagtca tttcccataa aatacagaca 1020ttttaccaac ataatatgct ttgattgatg cagcattatg ctttgggcag tattacaaaa 1080tagctggcga gtgctttctg tatttaaata ttgtaaaaag aaaataagtt ataactgtta 1140taaagcagaa cttttgttgc attttttaaa ctgttgaagt cnctgtgtat gtttgtttgg 1200tcaatgtttc cncagtattt attaaaacat actttttttt ttcttcaaat aaaaaagtaa 1260ccatgtcttt gtctaaaaaa aaaaaaanan nannnnaaaa aaa 1303 <210> SEQ ID NO 7<211> LENGTH: 244 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (118)..(118) <223>OTHER INFORMATION: “Xaa” is unknown <400> SEQUENCE: 7 Met Lys Glu ArgArg Ala Ser Gln Lys Leu Ser Ser Lys Ser Ile Met 1 5 10 15 Asp Pro AsnGln Asn Val Lys Cys Lys Ile Val Val Val Gly Asp Ser 20 25 30 Gln Cys GlyLys Thr Ala Leu Leu His Val Phe Ala Lys Asp Cys Phe 35 40 45 Pro Glu AsnTyr Val Pro Thr Val Phe Glu Asn Tyr Thr Ala Ser Phe 50 55 60 Glu Ile AspThr Gln Arg Ile Glu Leu Ser Leu Trp Asp Thr Ser Gly 65 70 75 80 Ser ProTyr Tyr Asp Asn Val Arg Pro Leu Ser Tyr Pro Asp Ser Asp 85 90 95 Ala ValLeu Ile Cys Phe Asp Ile Ser Arg Pro Glu Thr Leu Asp Ser 100 105 110 ValLeu Lys Lys Trp Xaa Gly Glu Ile Gln Glu Phe Cys Pro Asn Thr 115 120 125Lys Met Leu Leu Val Gly Cys Lys Ser Asp Leu Arg Thr Asp Val Ser 130 135140 Thr Leu Val Glu Leu Ser Asn His Arg Gln Thr Pro Val Ser Tyr Asp 145150 155 160 Gln Gly Ala Asn Met Ala Lys Gln Ile Gly Ala Ala Thr Tyr IleGlu 165 170 175 Cys Ser Ala Leu Gln Ser Glu Asn Ser Val Arg Asp Ile PheHis Val 180 185 190 Ala Thr Leu Ala Cys Val Asn Lys Thr Asn Lys Asn ValLys Arg Asn 195 200 205 Lys Ser Gln Arg Ala Thr Lys Arg Ile Ser His MetPro Ser Arg Pro 210 215 220 Glu Leu Ser Ala Val Ala Thr Asp Leu Arg LysAsp Lys Ala Lys Ser 225 230 235 240 Cys Thr Val Met <210> SEQ ID NO 8<211> LENGTH: 2479 <212> TYPE: DNA <213> ORGANISM: Rattus rattus <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(2274) <400> SEQUENCE:8 atg ggg gga tgc acg gtg aag cct cag ctg ctg ctc ctg gcg ctc gtc 48 MetGly Gly Cys Thr Val Lys Pro Gln Leu Leu Leu Leu Ala Leu Val 1 5 10 15ctc cac ccc tgg aat ccc tgt ctg ggt gcg gac tcg gag aag ccc tcg 96 LeuHis Pro Trp Asn Pro Cys Leu Gly Ala Asp Ser Glu Lys Pro Ser 20 25 30 agcatc ccc aca gat aaa tta tta gtc ata act gta gca aca aaa gaa 144 Ser IlePro Thr Asp Lys Leu Leu Val Ile Thr Val Ala Thr Lys Glu 35 40 45 agt gatgga ttc cat cga ttt atg cag tca gcc aaa tat ttc aat tat 192 Ser Asp GlyPhe His Arg Phe Met Gln Ser Ala Lys Tyr Phe Asn Tyr 50 55 60 act gtg aaggtc ctt ggt caa gga gaa gaa tgg aga ggt ggt gat gga 240 Thr Val Lys ValLeu Gly Gln Gly Glu Glu Trp Arg Gly Gly Asp Gly 65 70 75 80 att aat agtatt gga ggg ggc cag aaa gtg aga tta atg aaa gaa gtc 288 Ile Asn Ser IleGly Gly Gly Gln Lys Val Arg Leu Met Lys Glu Val 85 90 95 atg gaa cac tatgct gat caa gat gat ctg gtt gtc atg ttt act gaa 336 Met Glu His Tyr AlaAsp Gln Asp Asp Leu Val Val Met Phe Thr Glu 100 105 110 tgc ttt gat gtcata ttt gct ggt ggt cca gaa gaa gtt cta aaa aaa 384 Cys Phe Asp Val IlePhe Ala Gly Gly Pro Glu Glu Val Leu Lys Lys 115 120 125 ttc caa aag gcaaac cac aaa gtg gtc ttt gca gca gat gga att ttg 432 Phe Gln Lys Ala AsnHis Lys Val Val Phe Ala Ala Asp Gly Ile Leu 130 135 140 tgg cca gat aaaaga cta gca gac aag tat cct gtt gtg cac att ggg 480 Trp Pro Asp Lys ArgLeu Ala Asp Lys Tyr Pro Val Val His Ile Gly 145 150 155 160 aaa cgc tatctg aat tca gga gga ttt att ggc tat gct cca tat gtc 528 Lys Arg Tyr LeuAsn Ser Gly Gly Phe Ile Gly Tyr Ala Pro Tyr Val 165 170 175 aac cgt atagtt caa caa tgg aat ctc cag gat aat gat gat gat cag 576 Asn Arg Ile ValGln Gln Trp Asn Leu Gln Asp Asn Asp Asp Asp Gln 180 185 190 ctc ttt tacact aaa gtt tac att gat cca ctg aaa agg gaa gct att 624 Leu Phe Tyr ThrLys Val Tyr Ile Asp Pro Leu Lys Arg Glu Ala Ile 195 200 205 aac atc acattg gat cac aaa tgc aaa att ttc cag acc tta aat gga 672 Asn Ile Thr LeuAsp His Lys Cys Lys Ile Phe Gln Thr Leu Asn Gly 210 215 220 gct gta gatgaa gtt gtt tta aaa ttt gaa aat ggc aaa gcc aga gct 720 Ala Val Asp GluVal Val Leu Lys Phe Glu Asn Gly Lys Ala Arg Ala 225 230 235 240 aag aataca ttt tat gaa aca tta cca gtg gca att aat gga aat gga 768 Lys Asn ThrPhe Tyr Glu Thr Leu Pro Val Ala Ile Asn Gly Asn Gly 245 250 255 ccc accaag att ctc ctg aat tat ttt gga aac tat gta ccc aat tca 816 Pro Thr LysIle Leu Leu Asn Tyr Phe Gly Asn Tyr Val Pro Asn Ser 260 265 270 tgg acacag gat aat ggc tgc act ctt tgt gaa ttc gat aca gtc gac 864 Trp Thr GlnAsp Asn Gly Cys Thr Leu Cys Glu Phe Asp Thr Val Asp 275 280 285 ttg tctgca gta gat gtc cat cca aac gta tca ata ggt gtt ttt att 912 Leu Ser AlaVal Asp Val His Pro Asn Val Ser Ile Gly Val Phe Ile 290 295 300 gag caacca acc cct ttt cta cct cgg ttt ctg gac ata ttg ttg aca 960 Glu Gln ProThr Pro Phe Leu Pro Arg Phe Leu Asp Ile Leu Leu Thr 305 310 315 320 ctggat tac cca aaa gaa gca ctt aaa ctt ttt att cat aac aaa gaa 1008 Leu AspTyr Pro Lys Glu Ala Leu Lys Leu Phe Ile His Asn Lys Glu 325 330 335 gtttat cat gaa aag gac atc aag gta ttt ttt gat aaa gct aag cat 1056 Val TyrHis Glu Lys Asp Ile Lys Val Phe Phe Asp Lys Ala Lys His 340 345 350 gaaatc aaa act ata aaa ata gta gga cca gaa gaa aat cta agt caa 1104 Glu IleLys Thr Ile Lys Ile Val Gly Pro Glu Glu Asn Leu Ser Gln 355 360 365 gcggaa gcc aga aac atg gga atg gac ttt tgc cgt cag gat gaa aag 1152 Ala GluAla Arg Asn Met Gly Met Asp Phe Cys Arg Gln Asp Glu Lys 370 375 380 tgtgat tat tac ttt agt gtg gat gca gat gtt gtt ttg aca aat cca 1200 Cys AspTyr Tyr Phe Ser Val Asp Ala Asp Val Val Leu Thr Asn Pro 385 390 395 400agg act tta aaa att ttg att gaa caa aac aga aag atc att gct cct 1248 ArgThr Leu Lys Ile Leu Ile Glu Gln Asn Arg Lys Ile Ile Ala Pro 405 410 415ctt gta act cgt cat gga aag ctg tgg tcc aat ttc tgg gga gca ttg 1296 LeuVal Thr Arg His Gly Lys Leu Trp Ser Asn Phe Trp Gly Ala Leu 420 425 430agt cct gat gga tac tat gca cga tct gaa gat tat gtg gat att gtt 1344 SerPro Asp Gly Tyr Tyr Ala Arg Ser Glu Asp Tyr Val Asp Ile Val 435 440 445caa ggg aat aga gta gga gta tgg aat gtc cca tat atg gct aat gtg 1392 GlnGly Asn Arg Val Gly Val Trp Asn Val Pro Tyr Met Ala Asn Val 450 455 460tac tta att aaa gga aag aca ctc cga tca gag atg aat gaa agg aac 1440 TyrLeu Ile Lys Gly Lys Thr Leu Arg Ser Glu Met Asn Glu Arg Asn 465 470 475480 tat ttt gtt cgt gat aaa ctg gat cct gat atg gct ctt tgc cga aat 1488Tyr Phe Val Arg Asp Lys Leu Asp Pro Asp Met Ala Leu Cys Arg Asn 485 490495 gct aga gaa atg act tta caa agg gaa aaa gac tcc cct act ccg gaa 1536Ala Arg Glu Met Thr Leu Gln Arg Glu Lys Asp Ser Pro Thr Pro Glu 500 505510 aca ttc caa atg ctc agc ccc cca aag ggt gta ttt atg tac att tct 1584Thr Phe Gln Met Leu Ser Pro Pro Lys Gly Val Phe Met Tyr Ile Ser 515 520525 aat aga cat gaa ttt gga agg cta tta tcc act gct aat tac aat act 1632Asn Arg His Glu Phe Gly Arg Leu Leu Ser Thr Ala Asn Tyr Asn Thr 530 535540 tcc cat tat aac aat gac ctc tgg cag att ttt gaa aat cct gtg gac 1680Ser His Tyr Asn Asn Asp Leu Trp Gln Ile Phe Glu Asn Pro Val Asp 545 550555 560 tgg aag gaa aag tat ata aac cgt gat tat tca aag att ttc act gaa1728 Trp Lys Glu Lys Tyr Ile Asn Arg Asp Tyr Ser Lys Ile Phe Thr Glu 565570 575 aat ata gtt gaa cag ccc tgt cca gat gtc ttt tgg ttc ccc ata ttt1776 Asn Ile Val Glu Gln Pro Cys Pro Asp Val Phe Trp Phe Pro Ile Phe 580585 590 tct gaa aaa gcc tgt gat gaa ttg gta gaa gaa atg gaa cat tac ggc1824 Ser Glu Lys Ala Cys Asp Glu Leu Val Glu Glu Met Glu His Tyr Gly 595600 605 aaa tgg tct ggg gga aaa cat cat gat agc cgt ata tct ggt ggt tat1872 Lys Trp Ser Gly Gly Lys His His Asp Ser Arg Ile Ser Gly Gly Tyr 610615 620 gaa aat gtc cca act gat gat atc cac atg aag caa gtt gat ctg gag1920 Glu Asn Val Pro Thr Asp Asp Ile His Met Lys Gln Val Asp Leu Glu 625630 635 640 aat gta tgg ctt cat ttt atc cgg gag ttc att gca cca gtt acactg 1968 Asn Val Trp Leu His Phe Ile Arg Glu Phe Ile Ala Pro Val Thr Leu645 650 655 aag gtc ttt gca ggc tat tat acg aag gga ttt gca cta ctg aatttt 2016 Lys Val Phe Ala Gly Tyr Tyr Thr Lys Gly Phe Ala Leu Leu Asn Phe660 665 670 gta gta aaa tac tcc cct gaa cga cag cgt tct ctt cgt cct catcat 2064 Val Val Lys Tyr Ser Pro Glu Arg Gln Arg Ser Leu Arg Pro His His675 680 685 gat gct tct aca ttt acc ata aac att gca ctt aat aac gtg ggagaa 2112 Asp Ala Ser Thr Phe Thr Ile Asn Ile Ala Leu Asn Asn Val Gly Glu690 695 700 gac ttt cag gga ggt ggt tgc aaa ttt cta agg tac aat tgc tctatt 2160 Asp Phe Gln Gly Gly Gly Cys Lys Phe Leu Arg Tyr Asn Cys Ser Ile705 710 715 720 gag tca cca cga aaa ggc tgg agc ttc atg cat cct ggg agactc aca 2208 Glu Ser Pro Arg Lys Gly Trp Ser Phe Met His Pro Gly Arg LeuThr 725 730 735 cat ttg cat gaa gga ctt cct gtt aaa aat gga aca aga tacatt gca 2256 His Leu His Glu Gly Leu Pro Val Lys Asn Gly Thr Arg Tyr IleAla 740 745 750 gtg tca ttt ata gat ccc taagttattt acttttcatt gaattgaaat2304 Val Ser Phe Ile Asp Pro 755 ttattttgga tgaatgactg gcatgaacacgtctttgaag ttgtggctga gaagatgaga 2364 ggaatattta aataacatca acagaacaacttcactttgg gccaaacatt tgaaaaactt 2424 tttataaaaa attgtttgat atttcttaatgtctgctctg agccttaaaa cacag 2479 <210> SEQ ID NO 9 <211> LENGTH: 758<212> TYPE: PRT <213> ORGANISM: Rattus rattus <400> SEQUENCE: 9 Met GlyGly Cys Thr Val Lys Pro Gln Leu Leu Leu Leu Ala Leu Val 1 5 10 15 LeuHis Pro Trp Asn Pro Cys Leu Gly Ala Asp Ser Glu Lys Pro Ser 20 25 30 SerIle Pro Thr Asp Lys Leu Leu Val Ile Thr Val Ala Thr Lys Glu 35 40 45 SerAsp Gly Phe His Arg Phe Met Gln Ser Ala Lys Tyr Phe Asn Tyr 50 55 60 ThrVal Lys Val Leu Gly Gln Gly Glu Glu Trp Arg Gly Gly Asp Gly 65 70 75 80Ile Asn Ser Ile Gly Gly Gly Gln Lys Val Arg Leu Met Lys Glu Val 85 90 95Met Glu His Tyr Ala Asp Gln Asp Asp Leu Val Val Met Phe Thr Glu 100 105110 Cys Phe Asp Val Ile Phe Ala Gly Gly Pro Glu Glu Val Leu Lys Lys 115120 125 Phe Gln Lys Ala Asn His Lys Val Val Phe Ala Ala Asp Gly Ile Leu130 135 140 Trp Pro Asp Lys Arg Leu Ala Asp Lys Tyr Pro Val Val His IleGly 145 150 155 160 Lys Arg Tyr Leu Asn Ser Gly Gly Phe Ile Gly Tyr AlaPro Tyr Val 165 170 175 Asn Arg Ile Val Gln Gln Trp Asn Leu Gln Asp AsnAsp Asp Asp Gln 180 185 190 Leu Phe Tyr Thr Lys Val Tyr Ile Asp Pro LeuLys Arg Glu Ala Ile 195 200 205 Asn Ile Thr Leu Asp His Lys Cys Lys IlePhe Gln Thr Leu Asn Gly 210 215 220 Ala Val Asp Glu Val Val Leu Lys PheGlu Asn Gly Lys Ala Arg Ala 225 230 235 240 Lys Asn Thr Phe Tyr Glu ThrLeu Pro Val Ala Ile Asn Gly Asn Gly 245 250 255 Pro Thr Lys Ile Leu LeuAsn Tyr Phe Gly Asn Tyr Val Pro Asn Ser 260 265 270 Trp Thr Gln Asp AsnGly Cys Thr Leu Cys Glu Phe Asp Thr Val Asp 275 280 285 Leu Ser Ala ValAsp Val His Pro Asn Val Ser Ile Gly Val Phe Ile 290 295 300 Glu Gln ProThr Pro Phe Leu Pro Arg Phe Leu Asp Ile Leu Leu Thr 305 310 315 320 LeuAsp Tyr Pro Lys Glu Ala Leu Lys Leu Phe Ile His Asn Lys Glu 325 330 335Val Tyr His Glu Lys Asp Ile Lys Val Phe Phe Asp Lys Ala Lys His 340 345350 Glu Ile Lys Thr Ile Lys Ile Val Gly Pro Glu Glu Asn Leu Ser Gln 355360 365 Ala Glu Ala Arg Asn Met Gly Met Asp Phe Cys Arg Gln Asp Glu Lys370 375 380 Cys Asp Tyr Tyr Phe Ser Val Asp Ala Asp Val Val Leu Thr AsnPro 385 390 395 400 Arg Thr Leu Lys Ile Leu Ile Glu Gln Asn Arg Lys IleIle Ala Pro 405 410 415 Leu Val Thr Arg His Gly Lys Leu Trp Ser Asn PheTrp Gly Ala Leu 420 425 430 Ser Pro Asp Gly Tyr Tyr Ala Arg Ser Glu AspTyr Val Asp Ile Val 435 440 445 Gln Gly Asn Arg Val Gly Val Trp Asn ValPro Tyr Met Ala Asn Val 450 455 460 Tyr Leu Ile Lys Gly Lys Thr Leu ArgSer Glu Met Asn Glu Arg Asn 465 470 475 480 Tyr Phe Val Arg Asp Lys LeuAsp Pro Asp Met Ala Leu Cys Arg Asn 485 490 495 Ala Arg Glu Met Thr LeuGln Arg Glu Lys Asp Ser Pro Thr Pro Glu 500 505 510 Thr Phe Gln Met LeuSer Pro Pro Lys Gly Val Phe Met Tyr Ile Ser 515 520 525 Asn Arg His GluPhe Gly Arg Leu Leu Ser Thr Ala Asn Tyr Asn Thr 530 535 540 Ser His TyrAsn Asn Asp Leu Trp Gln Ile Phe Glu Asn Pro Val Asp 545 550 555 560 TrpLys Glu Lys Tyr Ile Asn Arg Asp Tyr Ser Lys Ile Phe Thr Glu 565 570 575Asn Ile Val Glu Gln Pro Cys Pro Asp Val Phe Trp Phe Pro Ile Phe 580 585590 Ser Glu Lys Ala Cys Asp Glu Leu Val Glu Glu Met Glu His Tyr Gly 595600 605 Lys Trp Ser Gly Gly Lys His His Asp Ser Arg Ile Ser Gly Gly Tyr610 615 620 Glu Asn Val Pro Thr Asp Asp Ile His Met Lys Gln Val Asp LeuGlu 625 630 635 640 Asn Val Trp Leu His Phe Ile Arg Glu Phe Ile Ala ProVal Thr Leu 645 650 655 Lys Val Phe Ala Gly Tyr Tyr Thr Lys Gly Phe AlaLeu Leu Asn Phe 660 665 670 Val Val Lys Tyr Ser Pro Glu Arg Gln Arg SerLeu Arg Pro His His 675 680 685 Asp Ala Ser Thr Phe Thr Ile Asn Ile AlaLeu Asn Asn Val Gly Glu 690 695 700 Asp Phe Gln Gly Gly Gly Cys Lys PheLeu Arg Tyr Asn Cys Ser Ile 705 710 715 720 Glu Ser Pro Arg Lys Gly TrpSer Phe Met His Pro Gly Arg Leu Thr 725 730 735 His Leu His Glu Gly LeuPro Val Lys Asn Gly Thr Arg Tyr Ile Ala 740 745 750 Val Ser Phe Ile AspPro 755 <210> SEQ ID NO 10 <211> LENGTH: 2479 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(2211) <400> SEQUENCE: 10 atg ggg gga tgc acg gtg aag cctcag ctg ctg ctc ctg gcg ctc gtc 48 Met Gly Gly Cys Thr Val Lys Pro GlnLeu Leu Leu Leu Ala Leu Val 1 5 10 15 ctc cac ccc tgg aat ccc tgt ctgggt gcg gac tcg gag aag ccc tcg 96 Leu His Pro Trp Asn Pro Cys Leu GlyAla Asp Ser Glu Lys Pro Ser 20 25 30 agc atc ccc aca gat aaa tta tta gtcata act gta gca aca aaa gaa 144 Ser Ile Pro Thr Asp Lys Leu Leu Val IleThr Val Ala Thr Lys Glu 35 40 45 agt gat gga ttc cat cga ttt atg cag tcagcc aaa tat ttc aat tat 192 Ser Asp Gly Phe His Arg Phe Met Gln Ser AlaLys Tyr Phe Asn Tyr 50 55 60 act gtg aag gtc ctt ggt caa gga gaa gaa tggaga ggt ggt gat gga 240 Thr Val Lys Val Leu Gly Gln Gly Glu Glu Trp ArgGly Gly Asp Gly 65 70 75 80 att aat agt att gga ggg ggc cag aaa gtg agatta atg aaa gaa gtc 288 Ile Asn Ser Ile Gly Gly Gly Gln Lys Val Arg LeuMet Lys Glu Val 85 90 95 atg gaa cac tat gct gat caa gat gat ctg gtt gtcatg ttt act gaa 336 Met Glu His Tyr Ala Asp Gln Asp Asp Leu Val Val MetPhe Thr Glu 100 105 110 tgc ttt gat gtc ata ttt gct ggt ggt cca gaa gaagtt cta aaa aaa 384 Cys Phe Asp Val Ile Phe Ala Gly Gly Pro Glu Glu ValLeu Lys Lys 115 120 125 ttc caa aag gca aac cac aaa gtg gtc ttt gca gcagat gga att ttg 432 Phe Gln Lys Ala Asn His Lys Val Val Phe Ala Ala AspGly Ile Leu 130 135 140 tgg cca gat aaa aga cta gca gac aag tat cct gttgtg cac att ggg 480 Trp Pro Asp Lys Arg Leu Ala Asp Lys Tyr Pro Val ValHis Ile Gly 145 150 155 160 aaa cgc tat ctg aat tca gga gga ttt att ggctat gct cca tat gtc 528 Lys Arg Tyr Leu Asn Ser Gly Gly Phe Ile Gly TyrAla Pro Tyr Val 165 170 175 aac cgt ata gtt caa caa tgg aat ctc cag gataat gat gat gat cag 576 Asn Arg Ile Val Gln Gln Trp Asn Leu Gln Asp AsnAsp Asp Asp Gln 180 185 190 ctc ttt tac act aaa gtt tac att gat cca ctgaaa agg gaa gct att 624 Leu Phe Tyr Thr Lys Val Tyr Ile Asp Pro Leu LysArg Glu Ala Ile 195 200 205 aac atc aca ttg gat cac aaa tgc aaa att ttccag acc tta aat gga 672 Asn Ile Thr Leu Asp His Lys Cys Lys Ile Phe GlnThr Leu Asn Gly 210 215 220 gct gta gat gaa gtt gtt tta aaa ttt gaa aatggc aaa gcc aga gct 720 Ala Val Asp Glu Val Val Leu Lys Phe Glu Asn GlyLys Ala Arg Ala 225 230 235 240 aag aat aca ttt tat gaa aca tta cca gtggca att aat gga aat gga 768 Lys Asn Thr Phe Tyr Glu Thr Leu Pro Val AlaIle Asn Gly Asn Gly 245 250 255 ccc acc aag att ctc ctg aat tat ttt ggaaac tat gta ccc aat tca 816 Pro Thr Lys Ile Leu Leu Asn Tyr Phe Gly AsnTyr Val Pro Asn Ser 260 265 270 tgg aca cag gat aat ggc tgc act ctt tgtgaa ttc gat aca gtc gac 864 Trp Thr Gln Asp Asn Gly Cys Thr Leu Cys GluPhe Asp Thr Val Asp 275 280 285 ttg tct gca gta gat gtc cat cca aac gtatca ata ggt gtt ttt att 912 Leu Ser Ala Val Asp Val His Pro Asn Val SerIle Gly Val Phe Ile 290 295 300 gag caa cca acc cct ttt cta cct cgg tttctg gac ata ttg ttg aca 960 Glu Gln Pro Thr Pro Phe Leu Pro Arg Phe LeuAsp Ile Leu Leu Thr 305 310 315 320 ctg gat tac cca aaa gaa gca ctt aaactt ttt att cat aac aaa gaa 1008 Leu Asp Tyr Pro Lys Glu Ala Leu Lys LeuPhe Ile His Asn Lys Glu 325 330 335 gtt tat cat gaa aag gac atc aag gtattt ttt gat aaa gct aag cat 1056 Val Tyr His Glu Lys Asp Ile Lys Val PhePhe Asp Lys Ala Lys His 340 345 350 gaa atc aaa act ata aaa ata gta ggacca gaa gaa aat cta agt caa 1104 Glu Ile Lys Thr Ile Lys Ile Val Gly ProGlu Glu Asn Leu Ser Gln 355 360 365 gcg gaa gcc aga aac atg gga atg gacttt tgc cgt cag gat gaa aag 1152 Ala Glu Ala Arg Asn Met Gly Met Asp PheCys Arg Gln Asp Glu Lys 370 375 380 tgt gat tat tac ttt agt gtg gat gcagat gtt gtt ttg aca aat cca 1200 Cys Asp Tyr Tyr Phe Ser Val Asp Ala AspVal Val Leu Thr Asn Pro 385 390 395 400 agg act tta aaa att ttg att gaacaa aac aga aag atc att gct cct 1248 Arg Thr Leu Lys Ile Leu Ile Glu GlnAsn Arg Lys Ile Ile Ala Pro 405 410 415 ctt gta act cgt cat gga aag ctgtgg tcc aat ttc tgg gga gca ttg 1296 Leu Val Thr Arg His Gly Lys Leu TrpSer Asn Phe Trp Gly Ala Leu 420 425 430 agt cct gat gga tac tat gca cgatct gaa gat tat gtg gat att gtt 1344 Ser Pro Asp Gly Tyr Tyr Ala Arg SerGlu Asp Tyr Val Asp Ile Val 435 440 445 caa ggg aat aga gta gga gta tggaat gtc cca tat atg gct aat gtg 1392 Gln Gly Asn Arg Val Gly Val Trp AsnVal Pro Tyr Met Ala Asn Val 450 455 460 tac tta att aaa gga aag aca ctccga tca gag atg aat gaa agg aac 1440 Tyr Leu Ile Lys Gly Lys Thr Leu ArgSer Glu Met Asn Glu Arg Asn 465 470 475 480 tat ttt gtt cgt gat aaa ctggat cct gat atg gct ctt tgc cga aat 1488 Tyr Phe Val Arg Asp Lys Leu AspPro Asp Met Ala Leu Cys Arg Asn 485 490 495 gct aga gaa atg act tta caaagg gaa aaa gac tcc cct act ccg gaa 1536 Ala Arg Glu Met Thr Leu Gln ArgGlu Lys Asp Ser Pro Thr Pro Glu 500 505 510 aca ttc caa atg ctc agc ccccca aag ggt gta ttt atg tac att tct 1584 Thr Phe Gln Met Leu Ser Pro ProLys Gly Val Phe Met Tyr Ile Ser 515 520 525 aat aga cat gaa ttt gga aggcta tta tcc act gct aat tac aat act 1632 Asn Arg His Glu Phe Gly Arg LeuLeu Ser Thr Ala Asn Tyr Asn Thr 530 535 540 tcc cat tat aac aat gac ctctgg cag att ttt gaa aat cct gtg gac 1680 Ser His Tyr Asn Asn Asp Leu TrpGln Ile Phe Glu Asn Pro Val Asp 545 550 555 560 tgg aag gaa aag tat ataaac cgt gat tat tca aag att ttc act gaa 1728 Trp Lys Glu Lys Tyr Ile AsnArg Asp Tyr Ser Lys Ile Phe Thr Glu 565 570 575 aat ata gtt gaa cag ccctgt cca gat gtc ttt tgg ttc ccc ata ttt 1776 Asn Ile Val Glu Gln Pro CysPro Asp Val Phe Trp Phe Pro Ile Phe 580 585 590 tct gaa aaa gcc tgt gatgaa ttg gta gaa gaa atg gaa cat tac ggc 1824 Ser Glu Lys Ala Cys Asp GluLeu Val Glu Glu Met Glu His Tyr Gly 595 600 605 aaa tgg tct ggg gga aaacat cat gat agc cgt ata tct ggt ggt tat 1872 Lys Trp Ser Gly Gly Lys HisHis Asp Ser Arg Ile Ser Gly Gly Tyr 610 615 620 gaa aat gtc cca act gatgat atc cac atg aag caa gtt gat ctg gag 1920 Glu Asn Val Pro Thr Asp AspIle His Met Lys Gln Val Asp Leu Glu 625 630 635 640 aat gta tgg ctt catttt atc cgg gag ttc att gca cca gtt aca ctg 1968 Asn Val Trp Leu His PheIle Arg Glu Phe Ile Ala Pro Val Thr Leu 645 650 655 aag gtc ttt gca ggctat tat acg aag gga ttt gca cta ctg aat ttt 2016 Lys Val Phe Ala Gly TyrTyr Thr Lys Gly Phe Ala Leu Leu Asn Phe 660 665 670 gta gta aaa tac tcccct gaa cga cag cgt tct ctt cgt cct cat cat 2064 Val Val Lys Tyr Ser ProGlu Arg Gln Arg Ser Leu Arg Pro His His 675 680 685 gat gct tct aca tttacc ata aac att gca ctt aat aac gtg gga gaa 2112 Asp Ala Ser Thr Phe ThrIle Asn Ile Ala Leu Asn Asn Val Gly Glu 690 695 700 gac ttt cag gga ggtggt tgc aaa ttt cta agg tac aat tgc tct att 2160 Asp Phe Gln Gly Gly GlyCys Lys Phe Leu Arg Tyr Asn Cys Ser Ile 705 710 715 720 gag tca cca cgaaaa ggc tgg agc ttc atg cat cct ggg aga ctc aca 2208 Glu Ser Pro Arg LysGly Trp Ser Phe Met His Pro Gly Arg Leu Thr 725 730 735 cat ttgcatgaaggacttcctgt taaaaatgga acaagataca ttgcagtgtc 2261 His atttatagatccctaagtta tttacttttc attgaattga aatttatttt ggatgaatga 2321 ctggcatgaacacgtctttg aagttgtggc tgagaagatg agaggaatat ttaaataaca 2381 tcaacagaacaacttcactt tgggccaaac atttgaaaaa ctttttataa aaaattgttt 2441 gatatttcttaatgtctgct ctgagcctta aaacacag 2479 <210> SEQ ID NO 11 <211> LENGTH: 737<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 11 Met GlyGly Cys Thr Val Lys Pro Gln Leu Leu Leu Leu Ala Leu Val 1 5 10 15 LeuHis Pro Trp Asn Pro Cys Leu Gly Ala Asp Ser Glu Lys Pro Ser 20 25 30 SerIle Pro Thr Asp Lys Leu Leu Val Ile Thr Val Ala Thr Lys Glu 35 40 45 SerAsp Gly Phe His Arg Phe Met Gln Ser Ala Lys Tyr Phe Asn Tyr 50 55 60 ThrVal Lys Val Leu Gly Gln Gly Glu Glu Trp Arg Gly Gly Asp Gly 65 70 75 80Ile Asn Ser Ile Gly Gly Gly Gln Lys Val Arg Leu Met Lys Glu Val 85 90 95Met Glu His Tyr Ala Asp Gln Asp Asp Leu Val Val Met Phe Thr Glu 100 105110 Cys Phe Asp Val Ile Phe Ala Gly Gly Pro Glu Glu Val Leu Lys Lys 115120 125 Phe Gln Lys Ala Asn His Lys Val Val Phe Ala Ala Asp Gly Ile Leu130 135 140 Trp Pro Asp Lys Arg Leu Ala Asp Lys Tyr Pro Val Val His IleGly 145 150 155 160 Lys Arg Tyr Leu Asn Ser Gly Gly Phe Ile Gly Tyr AlaPro Tyr Val 165 170 175 Asn Arg Ile Val Gln Gln Trp Asn Leu Gln Asp AsnAsp Asp Asp Gln 180 185 190 Leu Phe Tyr Thr Lys Val Tyr Ile Asp Pro LeuLys Arg Glu Ala Ile 195 200 205 Asn Ile Thr Leu Asp His Lys Cys Lys IlePhe Gln Thr Leu Asn Gly 210 215 220 Ala Val Asp Glu Val Val Leu Lys PheGlu Asn Gly Lys Ala Arg Ala 225 230 235 240 Lys Asn Thr Phe Tyr Glu ThrLeu Pro Val Ala Ile Asn Gly Asn Gly 245 250 255 Pro Thr Lys Ile Leu LeuAsn Tyr Phe Gly Asn Tyr Val Pro Asn Ser 260 265 270 Trp Thr Gln Asp AsnGly Cys Thr Leu Cys Glu Phe Asp Thr Val Asp 275 280 285 Leu Ser Ala ValAsp Val His Pro Asn Val Ser Ile Gly Val Phe Ile 290 295 300 Glu Gln ProThr Pro Phe Leu Pro Arg Phe Leu Asp Ile Leu Leu Thr 305 310 315 320 LeuAsp Tyr Pro Lys Glu Ala Leu Lys Leu Phe Ile His Asn Lys Glu 325 330 335Val Tyr His Glu Lys Asp Ile Lys Val Phe Phe Asp Lys Ala Lys His 340 345350 Glu Ile Lys Thr Ile Lys Ile Val Gly Pro Glu Glu Asn Leu Ser Gln 355360 365 Ala Glu Ala Arg Asn Met Gly Met Asp Phe Cys Arg Gln Asp Glu Lys370 375 380 Cys Asp Tyr Tyr Phe Ser Val Asp Ala Asp Val Val Leu Thr AsnPro 385 390 395 400 Arg Thr Leu Lys Ile Leu Ile Glu Gln Asn Arg Lys IleIle Ala Pro 405 410 415 Leu Val Thr Arg His Gly Lys Leu Trp Ser Asn PheTrp Gly Ala Leu 420 425 430 Ser Pro Asp Gly Tyr Tyr Ala Arg Ser Glu AspTyr Val Asp Ile Val 435 440 445 Gln Gly Asn Arg Val Gly Val Trp Asn ValPro Tyr Met Ala Asn Val 450 455 460 Tyr Leu Ile Lys Gly Lys Thr Leu ArgSer Glu Met Asn Glu Arg Asn 465 470 475 480 Tyr Phe Val Arg Asp Lys LeuAsp Pro Asp Met Ala Leu Cys Arg Asn 485 490 495 Ala Arg Glu Met Thr LeuGln Arg Glu Lys Asp Ser Pro Thr Pro Glu 500 505 510 Thr Phe Gln Met LeuSer Pro Pro Lys Gly Val Phe Met Tyr Ile Ser 515 520 525 Asn Arg His GluPhe Gly Arg Leu Leu Ser Thr Ala Asn Tyr Asn Thr 530 535 540 Ser His TyrAsn Asn Asp Leu Trp Gln Ile Phe Glu Asn Pro Val Asp 545 550 555 560 TrpLys Glu Lys Tyr Ile Asn Arg Asp Tyr Ser Lys Ile Phe Thr Glu 565 570 575Asn Ile Val Glu Gln Pro Cys Pro Asp Val Phe Trp Phe Pro Ile Phe 580 585590 Ser Glu Lys Ala Cys Asp Glu Leu Val Glu Glu Met Glu His Tyr Gly 595600 605 Lys Trp Ser Gly Gly Lys His His Asp Ser Arg Ile Ser Gly Gly Tyr610 615 620 Glu Asn Val Pro Thr Asp Asp Ile His Met Lys Gln Val Asp LeuGlu 625 630 635 640 Asn Val Trp Leu His Phe Ile Arg Glu Phe Ile Ala ProVal Thr Leu 645 650 655 Lys Val Phe Ala Gly Tyr Tyr Thr Lys Gly Phe AlaLeu Leu Asn Phe 660 665 670 Val Val Lys Tyr Ser Pro Glu Arg Gln Arg SerLeu Arg Pro His His 675 680 685 Asp Ala Ser Thr Phe Thr Ile Asn Ile AlaLeu Asn Asn Val Gly Glu 690 695 700 Asp Phe Gln Gly Gly Gly Cys Lys PheLeu Arg Tyr Asn Cys Ser Ile 705 710 715 720 Glu Ser Pro Arg Lys Gly TrpSer Phe Met His Pro Gly Arg Leu Thr 725 730 735 His <210> SEQ ID NO 12<211> LENGTH: 3718 <212> TYPE: DNA <213> ORGANISM: Rattus rattus <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (222)..(2486) <400>SEQUENCE: 12 ggctgcgaga agacgacaga aggggtccgt cgtctgctcg gtgcgctcgggctccgcgct 60 agtccgctca gtgttctcca atcgctttgg tacccacgca gtcctctcatccgtcctccg 120 ctgccgtccc gggccccacg tctaacccgg tgctcttcgg ggtctccgcgtctcgcgaga 180 agtcctcgcc gcaggcctcg ggctttcggg cttaggggcg g atg ggg gaccgc gga 236 Met Gly Asp Arg Gly 1 5 gtg agg ctg ggg ctg ctg atg ccc atgctc gcc ctg ctc tcc tgg gcg 284 Val Arg Leu Gly Leu Leu Met Pro Met LeuAla Leu Leu Ser Trp Ala 10 15 20 gct agc ctg ggc gta gcg gag gag act ccctcg cgc atc cca gca gat 332 Ala Ser Leu Gly Val Ala Glu Glu Thr Pro SerArg Ile Pro Ala Asp 25 30 35 aag tta tta gtc ata act gta gca acc aaa gaaaac gat gga ttc cac 380 Lys Leu Leu Val Ile Thr Val Ala Thr Lys Glu AsnAsp Gly Phe His 40 45 50 aga ttt atg aat tca gcc aag tat ttc aat tat actgtg aag gtt ctt 428 Arg Phe Met Asn Ser Ala Lys Tyr Phe Asn Tyr Thr ValLys Val Leu 55 60 65 ggt caa ggg caa gag tgg aga ggt ggt gat ggg atg aacagt att gga 476 Gly Gln Gly Gln Glu Trp Arg Gly Gly Asp Gly Met Asn SerIle Gly 70 75 80 85 ggg ggc cag aag gtg aga tta atg aaa gaa gcc atg gagcac tac gcc 524 Gly Gly Gln Lys Val Arg Leu Met Lys Glu Ala Met Glu HisTyr Ala 90 95 100 ggt cag gac gat ctg gtc atc ttg ttt act gaa tgt tttgat gtt ata 572 Gly Gln Asp Asp Leu Val Ile Leu Phe Thr Glu Cys Phe AspVal Ile 105 110 115 ttt gct ggt ggg cct gaa gaa ctt ctt aaa aag ttc caaaag aca aat 620 Phe Ala Gly Gly Pro Glu Glu Leu Leu Lys Lys Phe Gln LysThr Asn 120 125 130 cat aaa atc gtc ttt gca gcg gat gcg ctg ttg tgg ccagat aag cgg 668 His Lys Ile Val Phe Ala Ala Asp Ala Leu Leu Trp Pro AspLys Arg 135 140 145 ctg gca gac aag tat cct ggt gtg cac att ggg aaa cgctac ctg aat 716 Leu Ala Asp Lys Tyr Pro Gly Val His Ile Gly Lys Arg TyrLeu Asn 150 155 160 165 tct gga ggc ttt att ggc tat gct ccc tac atc agccgt ctg gtc cag 764 Ser Gly Gly Phe Ile Gly Tyr Ala Pro Tyr Ile Ser ArgLeu Val Gln 170 175 180 cag tgg gat ctg cag gat aat gat gac gac cag ctcttt tac act aaa 812 Gln Trp Asp Leu Gln Asp Asn Asp Asp Asp Gln Leu PheTyr Thr Lys 185 190 195 gtt tac atc gac ccg ctg aaa agg gaa gct ctt aacatc aca ttg gat 860 Val Tyr Ile Asp Pro Leu Lys Arg Glu Ala Leu Asn IleThr Leu Asp 200 205 210 cac aga tgc aaa att ttc cag gcc ttg aat gga gctaca gac gaa gtt 908 His Arg Cys Lys Ile Phe Gln Ala Leu Asn Gly Ala ThrAsp Glu Val 215 220 225 gtt tta aag ttt gaa aat ggt aaa agc aga gtg aagaat aca ttt tat 956 Val Leu Lys Phe Glu Asn Gly Lys Ser Arg Val Lys AsnThr Phe Tyr 230 235 240 245 gaa aca ctg cca gtg gcc atc aat ggg aat gggccc acc aaa att ctc 1004 Glu Thr Leu Pro Val Ala Ile Asn Gly Asn Gly ProThr Lys Ile Leu 250 255 260 ttg aat tac ttt gga aac tat gtt cca aat tcatgg aca cag gaa aat 1052 Leu Asn Tyr Phe Gly Asn Tyr Val Pro Asn Ser TrpThr Gln Glu Asn 265 270 275 ggc tgt gct ctt tgt gac ttt gac aca att gacctg tct aca gta gat 1100 Gly Cys Ala Leu Cys Asp Phe Asp Thr Ile Asp LeuSer Thr Val Asp 280 285 290 gtc tat ccg aag gta aca cta ggt gtt ttt attgaa caa cca acc ccc 1148 Val Tyr Pro Lys Val Thr Leu Gly Val Phe Ile GluGln Pro Thr Pro 295 300 305 ttt cta cct cgg ttc ctg gac tta ctg tta acactg gat tac cct aaa 1196 Phe Leu Pro Arg Phe Leu Asp Leu Leu Leu Thr LeuAsp Tyr Pro Lys 310 315 320 325 gaa gca ctt cga ctc ttt gtc cat aat aaagaa gtt tat cat gaa aag 1244 Glu Ala Leu Arg Leu Phe Val His Asn Lys GluVal Tyr His Glu Lys 330 335 340 gac atc aaa gcg ttt gtt gat aaa gct aaacac gac atc agc tct ata 1292 Asp Ile Lys Ala Phe Val Asp Lys Ala Lys HisAsp Ile Ser Ser Ile 345 350 355 aaa ata gta gga cca gag gaa aat cta agtcaa gcg gaa gcc aga aac 1340 Lys Ile Val Gly Pro Glu Glu Asn Leu Ser GlnAla Glu Ala Arg Asn 360 365 370 atg gga atg gat ttc tgc cgt cag gat gaaaag tgt gat tac tac ttt 1388 Met Gly Met Asp Phe Cys Arg Gln Asp Glu LysCys Asp Tyr Tyr Phe 375 380 385 agt gtg gat gca gat gtt gtt ttg aca aaccca aga act tta aaa att 1436 Ser Val Asp Ala Asp Val Val Leu Thr Asn ProArg Thr Leu Lys Ile 390 395 400 405 ttg att gaa caa aac agg aag atc attgcc cct ctt gtg aca cgt cat 1484 Leu Ile Glu Gln Asn Arg Lys Ile Ile AlaPro Leu Val Thr Arg His 410 415 420 gga aag ttg tgg tcc aac ttc tgg ggagcc ctg agt cct gat gga tac 1532 Gly Lys Leu Trp Ser Asn Phe Trp Gly AlaLeu Ser Pro Asp Gly Tyr 425 430 435 tat gct cgt tct gaa gat tac gta gatatc gtt cag gga aac aga gta 1580 Tyr Ala Arg Ser Glu Asp Tyr Val Asp IleVal Gln Gly Asn Arg Val 440 445 450 gga ata tgg aat gtc cca tac atg gctaat gtg tac tta att caa ggg 1628 Gly Ile Trp Asn Val Pro Tyr Met Ala AsnVal Tyr Leu Ile Gln Gly 455 460 465 aag acg ctg cga tca gag atg agt gaaagg aac tat ttt gtg cgt gat 1676 Lys Thr Leu Arg Ser Glu Met Ser Glu ArgAsn Tyr Phe Val Arg Asp 470 475 480 485 aag ttg gat ccc gac atg tct ctctgc cgc aat gct cga gac atg acc 1724 Lys Leu Asp Pro Asp Met Ser Leu CysArg Asn Ala Arg Asp Met Thr 490 495 500 tta caa agg gaa aaa gac tcc cccact ccg gaa aca ttc caa atg ctc 1772 Leu Gln Arg Glu Lys Asp Ser Pro ThrPro Glu Thr Phe Gln Met Leu 505 510 515 agc ccc cca aag ggt gtg ttt atgtac att tct aac aga cat gaa ttt 1820 Ser Pro Pro Lys Gly Val Phe Met TyrIle Ser Asn Arg His Glu Phe 520 525 530 gga cgg ctg ata tca act gct aattac aac act tcc cat ctc aac aat 1868 Gly Arg Leu Ile Ser Thr Ala Asn TyrAsn Thr Ser His Leu Asn Asn 535 540 545 gac ctc tgg cag atc ttt gaa aatccc gtg gat tgg aag gaa aaa tat 1916 Asp Leu Trp Gln Ile Phe Glu Asn ProVal Asp Trp Lys Glu Lys Tyr 550 555 560 565 ata aac cgt gac tat tca aagatt ttc act gaa aat ata gtc gag cag 1964 Ile Asn Arg Asp Tyr Ser Lys IlePhe Thr Glu Asn Ile Val Glu Gln 570 575 580 ccc tgt cca gat gtc ttc tggttt ccc ata ttt tct gaa cga gcc tgt 2012 Pro Cys Pro Asp Val Phe Trp PhePro Ile Phe Ser Glu Arg Ala Cys 585 590 595 gac gag ttg gta gaa gaa atggaa cat tac ggc aag tgg tcc ggg gga 2060 Asp Glu Leu Val Glu Glu Met GluHis Tyr Gly Lys Trp Ser Gly Gly 600 605 610 aag cat cat gac agc cgt atatct ggt ggc tat gaa aat gtc cca acg 2108 Lys His His Asp Ser Arg Ile SerGly Gly Tyr Glu Asn Val Pro Thr 615 620 625 gat gac att cat atg aag cagatt gac ctg gag aac gtc tgg ctt cac 2156 Asp Asp Ile His Met Lys Gln IleAsp Leu Glu Asn Val Trp Leu His 630 635 640 645 ttt atc cga gag ttt atcgct cca gtt acc ctg aag gtc ttc gcg gga 2204 Phe Ile Arg Glu Phe Ile AlaPro Val Thr Leu Lys Val Phe Ala Gly 650 655 660 tat tac acc aag gga tttgcc ctg ctg aac ttc gta gtg aag tac tcg 2252 Tyr Tyr Thr Lys Gly Phe AlaLeu Leu Asn Phe Val Val Lys Tyr Ser 665 670 675 ccc gaa aga cag cgc tcgctc cgg cct cac cac gat gcg tca acc ttc 2300 Pro Glu Arg Gln Arg Ser LeuArg Pro His His Asp Ala Ser Thr Phe 680 685 690 acc atc aac att gct ctaaat aat gta gga gag gat ttt cag gga ggt 2348 Thr Ile Asn Ile Ala Leu AsnAsn Val Gly Glu Asp Phe Gln Gly Gly 695 700 705 gga tgc aaa ttc cta aggtat aat tgc tcc atc gaa tcc ccc cga aaa 2396 Gly Cys Lys Phe Leu Arg TyrAsn Cys Ser Ile Glu Ser Pro Arg Lys 710 715 720 725 ggc tgg agc ttc atgcat cct ggg agg ctt act cat cta cac gaa ggg 2444 Gly Trp Ser Phe Met HisPro Gly Arg Leu Thr His Leu His Glu Gly 730 735 740 ctt cct gtc aaa aatgga aca aga tac att gca gtc tca ttt 2486 Leu Pro Val Lys Asn Gly Thr ArgTyr Ile Ala Val Ser Phe 745 750 755 atcgatccct aagttattga ctgaacttaaactgagtggc tctttgagat ggatgactgg 2546 cgggaacatg tctctgaagt tgtacttgagaagacgagag gaatatttaa ataatgtcac 2606 cagaacaacg tcactttggg ccaagcatttgaaaactttt tatataaatt tgttttatgt 2666 ttcttaacgt ctgctctgag ccttaaaacacaggttgaag aagaagagag aggaaaaaag 2726 tgaaagttgg tatttatttc tgtgctttaattgtctatga aaatgatgac attttataaa 2786 atgtttaggt acaaaggcat gaatgataatcagtaagcct aataatattt tcttatttaa 2846 ggagaacctg agaagatttt atttttcagtgggagaaata tggaaaatgg ttctaaatga 2906 gggtcggcac gtctggaagc ccgggattctgacgcgtact gaatttatgt gtaactttta 2966 agccatgctg acctccgggt agattcgcttttcagtgata aggaagaaaa cccaaagaaa 3026 atattgcaca gaggctttcc tcaagcagcctgggcagatg gccagtggaa gcccatccac 3086 tggagatcct cagcttgtga ggcaggtgctcctgtccgtt ggaaactggg cccctgtgtg 3146 tctccagggc aagctctcag gggaagctcacatctgcctg ctttacagag tgcttcaggc 3206 gtcagctcca agtcaaacag gatgtgtttccttctgtttt tcccctctaa ttatagaaaa 3266 tagtaaggaa aaatatcagt ttcattgagattagtagtac attttactat cttctttttt 3326 aacgattaag tacttgaatt ttatatcaggaaaatagttt ttgagcctgt tcttaccttt 3386 ggccgtagtt ggtagttggt ctctttgtttttcctggagg aggggcattt cttttcctca 3446 tcataaacta ctttctcatt cttagtcttgttattacttt tcctctaccc cactttttaa 3506 aaattcccac agcaaaattt ttatttgaatttttaatatt tctctgaatg aggtttaaat 3566 atctttatta gagctactgt ttttaatttaaaggttaaac ttgaagaaag tctttattca 3626 tggtgccaaa atgcattttt ctaactctgtgtgttagaaa ataatgaaaa ataaaataac 3686 ttacaataaa aaaaaaaaaa aaaaaaaaaaaa 3718 <210> SEQ ID NO 13 <211> LENGTH: 755 <212> TYPE: PRT <213>ORGANISM: Rattus rattus <400> SEQUENCE: 13 Met Gly Asp Arg Gly Val ArgLeu Gly Leu Leu Met Pro Met Leu Ala 1 5 10 15 Leu Leu Ser Trp Ala AlaSer Leu Gly Val Ala Glu Glu Thr Pro Ser 20 25 30 Arg Ile Pro Ala Asp LysLeu Leu Val Ile Thr Val Ala Thr Lys Glu 35 40 45 Asn Asp Gly Phe His ArgPhe Met Asn Ser Ala Lys Tyr Phe Asn Tyr 50 55 60 Thr Val Lys Val Leu GlyGln Gly Gln Glu Trp Arg Gly Gly Asp Gly 65 70 75 80 Met Asn Ser Ile GlyGly Gly Gln Lys Val Arg Leu Met Lys Glu Ala 85 90 95 Met Glu His Tyr AlaGly Gln Asp Asp Leu Val Ile Leu Phe Thr Glu 100 105 110 Cys Phe Asp ValIle Phe Ala Gly Gly Pro Glu Glu Leu Leu Lys Lys 115 120 125 Phe Gln LysThr Asn His Lys Ile Val Phe Ala Ala Asp Ala Leu Leu 130 135 140 Trp ProAsp Lys Arg Leu Ala Asp Lys Tyr Pro Gly Val His Ile Gly 145 150 155 160Lys Arg Tyr Leu Asn Ser Gly Gly Phe Ile Gly Tyr Ala Pro Tyr Ile 165 170175 Ser Arg Leu Val Gln Gln Trp Asp Leu Gln Asp Asn Asp Asp Asp Gln 180185 190 Leu Phe Tyr Thr Lys Val Tyr Ile Asp Pro Leu Lys Arg Glu Ala Leu195 200 205 Asn Ile Thr Leu Asp His Arg Cys Lys Ile Phe Gln Ala Leu AsnGly 210 215 220 Ala Thr Asp Glu Val Val Leu Lys Phe Glu Asn Gly Lys SerArg Val 225 230 235 240 Lys Asn Thr Phe Tyr Glu Thr Leu Pro Val Ala IleAsn Gly Asn Gly 245 250 255 Pro Thr Lys Ile Leu Leu Asn Tyr Phe Gly AsnTyr Val Pro Asn Ser 260 265 270 Trp Thr Gln Glu Asn Gly Cys Ala Leu CysAsp Phe Asp Thr Ile Asp 275 280 285 Leu Ser Thr Val Asp Val Tyr Pro LysVal Thr Leu Gly Val Phe Ile 290 295 300 Glu Gln Pro Thr Pro Phe Leu ProArg Phe Leu Asp Leu Leu Leu Thr 305 310 315 320 Leu Asp Tyr Pro Lys GluAla Leu Arg Leu Phe Val His Asn Lys Glu 325 330 335 Val Tyr His Glu LysAsp Ile Lys Ala Phe Val Asp Lys Ala Lys His 340 345 350 Asp Ile Ser SerIle Lys Ile Val Gly Pro Glu Glu Asn Leu Ser Gln 355 360 365 Ala Glu AlaArg Asn Met Gly Met Asp Phe Cys Arg Gln Asp Glu Lys 370 375 380 Cys AspTyr Tyr Phe Ser Val Asp Ala Asp Val Val Leu Thr Asn Pro 385 390 395 400Arg Thr Leu Lys Ile Leu Ile Glu Gln Asn Arg Lys Ile Ile Ala Pro 405 410415 Leu Val Thr Arg His Gly Lys Leu Trp Ser Asn Phe Trp Gly Ala Leu 420425 430 Ser Pro Asp Gly Tyr Tyr Ala Arg Ser Glu Asp Tyr Val Asp Ile Val435 440 445 Gln Gly Asn Arg Val Gly Ile Trp Asn Val Pro Tyr Met Ala AsnVal 450 455 460 Tyr Leu Ile Gln Gly Lys Thr Leu Arg Ser Glu Met Ser GluArg Asn 465 470 475 480 Tyr Phe Val Arg Asp Lys Leu Asp Pro Asp Met SerLeu Cys Arg Asn 485 490 495 Ala Arg Asp Met Thr Leu Gln Arg Glu Lys AspSer Pro Thr Pro Glu 500 505 510 Thr Phe Gln Met Leu Ser Pro Pro Lys GlyVal Phe Met Tyr Ile Ser 515 520 525 Asn Arg His Glu Phe Gly Arg Leu IleSer Thr Ala Asn Tyr Asn Thr 530 535 540 Ser His Leu Asn Asn Asp Leu TrpGln Ile Phe Glu Asn Pro Val Asp 545 550 555 560 Trp Lys Glu Lys Tyr IleAsn Arg Asp Tyr Ser Lys Ile Phe Thr Glu 565 570 575 Asn Ile Val Glu GlnPro Cys Pro Asp Val Phe Trp Phe Pro Ile Phe 580 585 590 Ser Glu Arg AlaCys Asp Glu Leu Val Glu Glu Met Glu His Tyr Gly 595 600 605 Lys Trp SerGly Gly Lys His His Asp Ser Arg Ile Ser Gly Gly Tyr 610 615 620 Glu AsnVal Pro Thr Asp Asp Ile His Met Lys Gln Ile Asp Leu Glu 625 630 635 640Asn Val Trp Leu His Phe Ile Arg Glu Phe Ile Ala Pro Val Thr Leu 645 650655 Lys Val Phe Ala Gly Tyr Tyr Thr Lys Gly Phe Ala Leu Leu Asn Phe 660665 670 Val Val Lys Tyr Ser Pro Glu Arg Gln Arg Ser Leu Arg Pro His His675 680 685 Asp Ala Ser Thr Phe Thr Ile Asn Ile Ala Leu Asn Asn Val GlyGlu 690 695 700 Asp Phe Gln Gly Gly Gly Cys Lys Phe Leu Arg Tyr Asn CysSer Ile 705 710 715 720 Glu Ser Pro Arg Lys Gly Trp Ser Phe Met His ProGly Arg Leu Thr 725 730 735 His Leu His Glu Gly Leu Pro Val Lys Asn GlyThr Arg Tyr Ile Ala 740 745 750 Val Ser Phe 755 <210> SEQ ID NO 14 <211>LENGTH: 1486 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(396) <400> SEQUENCE: 14 gctctg tac gcg gcg ctg gcc gcc ttg gag gag cac cgg cgg gtc agc 48 Ala LeuTyr Ala Ala Leu Ala Ala Leu Glu Glu His Arg Arg Val Ser 1 5 10 15 cacggt gag ggc ggc ggg gag gag gcg gcg gcc gcc gcc cgg gaa agg 96 His GlyGlu Gly Gly Gly Glu Glu Ala Ala Ala Ala Ala Arg Glu Arg 20 25 30 gga tcggcg tcc ggg gaa ccc ccg tct ggc tcc ggc cgc ggc aag aag 144 Gly Ser AlaSer Gly Glu Pro Pro Ser Gly Ser Gly Arg Gly Lys Lys 35 40 45 atc ttc ggctgc tcc gag tgc gag aag ctg ttc cgc tca ccg cga gac 192 Ile Phe Gly CysSer Glu Cys Glu Lys Leu Phe Arg Ser Pro Arg Asp 50 55 60 ctg gag cgg cacgtg ctg gtg cac act ggc gag aag ccg ttc ccg tgc 240 Leu Glu Arg His ValLeu Val His Thr Gly Glu Lys Pro Phe Pro Cys 65 70 75 80 ctg gag tgc ggcaag ttc ttc cgc cac gag tgc tac ctc aag cgc cac 288 Leu Glu Cys Gly LysPhe Phe Arg His Glu Cys Tyr Leu Lys Arg His 85 90 95 cga ctg ctg cac ggcacc gag cgg ccc ttc cct tgc cac atc tgc ggc 336 Arg Leu Leu His Gly ThrGlu Arg Pro Phe Pro Cys His Ile Cys Gly 100 105 110 aag ggc ttc atc acgctc agc aac ctc tcc agg cac ctg aag ctg cac 384 Lys Gly Phe Ile Thr LeuSer Asn Leu Ser Arg His Leu Lys Leu His 115 120 125 cgg ggc atg gactgactgccag gctgcgtgcg ccctgccctc cacccagcct 436 Arg Gly Met Asp 130cctggactcg gcctggacca ggggacctcg ggactgcgcg tgaggccccg gccctccaaa 496tccaaatcca gacgcaggcc ctgaaatgag gggaccctga ctggagaggt gggggccacc 556aaaaacccac aaaggccccg gagctggggg accacaaaca aacagggtcc ttagctgggg 616caggggagcc caaatctagg gagagactcc tgagcctgag gtccctggaa tgagtgtggg 676tagccgtaag tccccaagac atggggactt tgcagtgagc aatgggtctc cacaagtacc 736tctcatcttg agagccctaa tactaaaaga tgggcaccca cccccaccaa gggaagactg 796ccccattccc tgagagccat cattcctaac gaccttgatc tggagaatgt ggagggagca 856tgtccctgaa ttttcctaga tccctccaaa tgccacccac cagagtcact ggtgacccca 916gaaaatggat atagccgaaa tctgcctttc ccctttttca ttccctgtgc tgaaagaggg 976accagggtag atgccccctg ccctcgaatc ccccctcccc gactgtggaa tggatcgacc 1036ctaacgatct tccccgcccc aaacactaga atagactggc ctgaaatccc cttgcccagt 1096agaatggact gatctatgtg cacacacccc catcacatgg aatgggctgg tctaggctgt 1156ggcctgccac cttccttaga gtgaataggg gggacactcc tttttttttc ctgtagggtg 1216tgggccggtc cacgcaattt tttatcctgt gaactcattt gagtgggagg tggtggacac 1276ctggggtttc cttccctctc tccgtagcat ccgttggtct ttctctccat ctctgttggt 1336ttgtctgtct ctgtcttcct cccaatccct aggggaaggg ggcatttggc tagggggtgc 1396ccctgtgagc ctcgaccttg ccccctcgtc cctctcccca gtgtttccag gacccccaat 1456aaaccttgtc ctgtcaaaaa aaaaaaaaaa 1486 <210> SEQ ID NO 15 <211> LENGTH:132 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 15 AlaLeu Tyr Ala Ala Leu Ala Ala Leu Glu Glu His Arg Arg Val Ser 1 5 10 15His Gly Glu Gly Gly Gly Glu Glu Ala Ala Ala Ala Ala Arg Glu Arg 20 25 30Gly Ser Ala Ser Gly Glu Pro Pro Ser Gly Ser Gly Arg Gly Lys Lys 35 40 45Ile Phe Gly Cys Ser Glu Cys Glu Lys Leu Phe Arg Ser Pro Arg Asp 50 55 60Leu Glu Arg His Val Leu Val His Thr Gly Glu Lys Pro Phe Pro Cys 65 70 7580 Leu Glu Cys Gly Lys Phe Phe Arg His Glu Cys Tyr Leu Lys Arg His 85 9095 Arg Leu Leu His Gly Thr Glu Arg Pro Phe Pro Cys His Ile Cys Gly 100105 110 Lys Gly Phe Ile Thr Leu Ser Asn Leu Ser Arg His Leu Lys Leu His115 120 125 Arg Gly Met Asp 130 <210> SEQ ID NO 16 <211> LENGTH: 580<212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 16tttctttttt tctttcggcg tctgcggtgc tcggaagtgt ggtacttctc ctagttgcag 60tcaggcttca tacgctattg tcctgcccgt tagagcagcc agcgggtaca gaatggattt 120tggaagaggg agtcaccact ggacctccaa ggaagccacg tgcagacatc tacaaccttc 180gatctcctga cgagtttatt gttggccaaa accaggcttt gattgaacca ggatgaatgc 240gggtgttgga agtagaatat atatatacat ataaaattgg ttgggagcca cgtgtaccag 300tgtgtgttga tcttggcttg attcagtctg ccttgtaaca gaaactggcg atggaatatg 360agaggagccc tctggaaaga aaaggacaga ccctgtgctt tcatgaaagt gaagatctgg 420ctgaaccagt tccacaaggt tactgtatac atagcctgag tttaaaaggc tgtgcccact 480tcaagaatgt cattgttaga ctttgaaatt tctaactgcc tacctgcata aagaaaataa 540aatcttttaa atcaaaaaaa aaaaaaaaaa aaaaaaaaaa 580 <210> SEQ ID NO 17 <211>LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:17 Met Tyr Thr Val Thr Leu Trp Asn Trp Phe Ser Gln Ile Phe Thr Phe 1 510 15 Met Lys Ala Gln Gly Leu Ser Phe Ser Phe Gln Arg Ala Pro Leu Ile 2025 30 Phe His Arg Gln Phe Leu Leu Gln Gly Arg Leu Asn Gln Ala Lys Ile 3540 45 Asn Thr His Trp Tyr Thr Trp Leu Pro Thr Asn Phe Ile Cys Ile Tyr 5055 60 Ile Phe Tyr Phe Gln His Pro His Ser Ser Trp Phe Asn Gln Ser Leu 6570 75 80 Val Leu Ala Asn Asn Lys Leu Val Arg Arg Ser Lys Val Val Asp Val85 90 95 Cys Thr Trp Leu Pro Trp Arg Ser Ser Gly Asp Ser Leu Phe Gln Asn100 105 110 Pro Phe Cys Thr Arg Trp Leu Leu 115 120 <210> SEQ ID NO 18<211> LENGTH: 4342 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 18 ggcacgagag gcacagcacg acctctatgc agacaagtga actgtagaaactgattactg 60 ctccaccaag aagcccccat aagagtggtt atcctggaca cagaagtgttgaattgaaat 120 ccacagagca ttttacaaga gttctgacct ggatggggta aacctcagtgcacttctttt 180 ctgttggcct cagtattact ggattgaaga attgctgctt cttgttaggaggttcatttc 240 acttatcatt acttacaact tcatactcaa agcactgaga atttcaagtggagtatattg 300 aagtagactt cagtttcttt gcatcatttc tgtattcaat ttttttaattatttcataac 360 cctattgagt gttttttaac taaattaaca tggctcgaat gaaccgcccagctcctgtgg 420 aagtcacata caagaacatg agatttctta ttacacacaa tccaaccaatgcgaccttaa 480 acaaatttat agaggaactt aagaagtatg gagttaccac aatagtaagagtatgtgaag 540 caacttatga cactactctt gtggagaaag aaggtatcca tgttcttgattggccttttg 600 atgatggtgc accaccatcc aaccagattg ttgatgactg ggtaagtcttgtgaaaatta 660 agtttcgtga agaacctggt tgttgtattg ctgttcattg cgttgcaggccttgggagag 720 ctccagtact tgttgcccta gcattaattg aaggtggaat gaaatacgaagatgcagtac 780 aattcataag acaaaagcgg cgtggagctt ttaacagcaa gcaacttctgtatttggaga 840 agtatcgtcc taaaatgcgg ctgcgtttca aagattccaa cggtcatagaaacaactgtt 900 gcattcaata aaattggggt gcctaatgct actggaagtg gaacttgagatagggcctaa 960 tttgttatac atattagcca acatgttggc ttagtaagtc taatgaagcttccataggag 1020 tattgaaagg cagttttacc aggcctcaag ctagacagat ttggcaacctctgtatttgg 1080 gttacagtca acctatttgg atacttggca aaagattctt gctgtcagcatataaaatgt 1140 gcttgtcatt tgtatcaatt gacctttccc caaatcatgc agtattgagttatgacttgt 1200 taaatctatt cccatgccag aatcttatca atacataaga aatttaggaagattaggtgc 1260 caaaataccc agcacaatac ttgtatattt ttagtaccat acagaagtaaaatcccagga 1320 actatgaaca ctagacctta tgtggtttat tccttcaatc atttcaaacattgaaagtag 1380 ggcctacatg gttatttgcc tgctcacttt atgtttacat ctcccacattcataccaata 1440 tacgtcaggt ttgcttaacc attgattttt ttttttttta ccaagtcttacagtgattat 1500 tttacgtgtt tccatgtatc tcactttgtg ctgtattaaa aaaacctccattttgaaaat 1560 ctacgttgta cagaagcaca tgtctttaat gtcttcagac aaaaaagccttacattaatt 1620 taatgtttgc actctgaggt gcaacttaac agggagggcc tgagaaaagaatgggagggg 1680 gctattaatt atttttagca aaatgttgcc tttgtcttgt gcaaacatgtagaatatgct 1740 ctttaattta gtaaaatatt tttttaaaag gtagagatgc tttggtattggtatcataaa 1800 cttcctgaaa ttcttgaatt tttttcccat actatcaaga agtgtgtttaccacttattt 1860 ttgtttgaaa gtgtgatttt ttttttcctt cccaacctct ccttgcaaaaaaagaaatgg 1920 gtttctgcta atgaattgag cagacatcta atattttata tgccttttgagctgtgtaac 1980 ttaatatttg gatacttgac aatttgtttt attatgtaat tgataaaatggtgatgtgta 2040 ttaatgttag ttcaaccata tatttatact gtctggggat gtgtggttatagttctgtgg 2100 gagaaataat tttgtcagtg ttcaccagct tgtaaaaact tagtgcgagagctgaaacat 2160 ctaaataaat aatgacatgc atttatcatc attgagattg gtttgcttaaaattaactta 2220 ttttgtagaa gacaaaatga attgcacttc acttaatgtg tgtcctcatctttttacaaa 2280 taaatgaagg attataaatg atgtcagcat tttagtaaac ttttagacaaaatttgttag 2340 ggtcattcat gaaaacttta atactaaaag cactttccat tatatactttttaaaggtct 2400 agataatttt gaaccaattt attattgtgt actgaggaga aataatgtatagtagaggac 2460 agccttggtt tgtaaagctc agttccacta gttcatggtt ttgtgcaacttttgagcctc 2520 agttttctcc tttgcaaatt aataattaca tacctttata gattttgaaattaatttaaa 2580 tattagtatt tggacatgaa ggcttaatgt taagtttcct ttaatgatccacaataatcc 2640 ctttgatcac gttaatctaa atctagatgt ctttgtctaa ttttttttgaatagcagtta 2700 taaatgtaaa ggactcaaag tttaagtaaa aagtgatact ccaccttgtgtttcaaagaa 2760 tttagttcca cctcttcata ccagtttaac acttaatata tttcattggattttagacag 2820 ggcaaaagga agaacagggg cctctggagg cccttggtta tttaaatcttggattatttg 2880 tgatagtaat cacaaatttt tggctaattt ttaacctgag gttttgtttttttttttaaa 2940 ggaaatgcag cctagtcttg agaacataat tttatataat caattactaaatgttaaact 3000 attaccacac agcccataaa acagcatttg cgtttattga gagagaggatgtgccatcat 3060 gattaatgaa aactatcttt tgagtttgaa aagaaattaa tttgcagtgtttggattgta 3120 tatatggtgc taaaaataaa ttaatttact ttataaacct tatctgtacattatacgatg 3180 tgatgaaatt tgctttttat ccaaatattt tgtatcttgt aaatatggctaattatagga 3240 atgcctataa tacatcttag attccttata tctaataaga gttcaaagagttatgagttg 3300 aagtcttgaa tgcaggaaac tatctgatag tgttctaaaa tttggttacttgggtttgga 3360 tacccttagt gggatgatgt aaatagaggc tagctaccta ggcttgtctatagcaaccat 3420 aatgttgatg taagtaatgc ggttactgaa tcataagaaa atgccatctctttttagttg 3480 aaggaaaact ctggaagtag gtgccattgg tcattctgca gtgcactgcaaccattgttt 3540 cccctagtgc cctcttttcc ctagggcatt gctctcctat tcccacgccttaacacagct 3600 ctatacctag aagcagccag cccaggcatg cagtcacatt taatcacatcccccttctag 3660 agtgcttcaa aatgatgtag tccctcaact tggctaaaga atctcaatctcttgaaattt 3720 atttttttaa tgtcatattc atctggtaaa tatctactgt ttgccaggcatttaagaata 3780 tggcaaagaa cataaaagat ggtgtcacca gattttggtc accaatgagtacccgacccg 3840 ttgccatgat taagagagaa tgctttctat tggagtttca ggaaatataatttgagaata 3900 ctttaaaggg aagtggaagt ataagtgaat gatatttttc ttttacatgtaaacaatgaa 3960 gttatttcaa agttaagttt taaacaaaat ccatgaagta gtgtctgccatacatgttaa 4020 tattctacat tcttgcttcc cttaaattaa tatgtttgtg tgtatatatgtgcctcacac 4080 ctgaattgaa aattaaagac tggtttaaaa gtggtttaaa agtgacatttaatgtttctc 4140 cattacgttt ggggtaacca gcctaagtgg aatcttggaa ggaaagtaagggaaaaactt 4200 gtatttgcct tcaatgaatt aaaccagtga tatgtttaac gtatgaatgaaaggattgat 4260 ggtgatttta taattatata tattgccgca gtaaccagtt aataaattgatagctaccat 4320 ttaaaaaaaa aaaaaaaaaa aa 4342 <210> SEQ ID NO 19 <211>LENGTH: 960 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:19 gccaagaatt cggcacgagg agtgggtatc tgagttagtc agttttacca tttataaaat 60gtttggtgga ggaaattgaa actaattgct aaattgttag taaccagtgc attaactagg 120accctactga gtggactgaa agaatcgaaa atgtttaact ggttgagagg caatgatgtt 180gcaaatgggg tattcttcaa agctccttct ttttttaaat cttcaaaggc aattattctg 240aatgtaaact acagaccaaa ttgcagtctt ctgtaagcat ttcagagatt acctcaaata 300ttttttgatt aaaaaactct tccgtggtct tttgtgcttc agaactaccc agtacaacag 360ggtcttcagc ctgctcagga tctctaaaga gagctagcac acagtcagcc aactttggct 420gcttcaactc ctaggaacaa gaaatgatgc tgagataatt tgtctggcag gtattatcag 480cccacaatga ctgctgtcat ttagcctcaa aatgtttatt ttttttttta caatgctgta 540tttctttaga accttcctat tccgagtgtg gaccctaggc cagccccata gacttcccct 600ggggacttgt cagaaatgca taattttagg ccccacccca gacctgttgg accagaatct 660tcatttaaca agatgcccag gtgattcatt catgtttgag aagctctgct ttaaatcact 720aaagcagtta ctgagtaatt actaccatca tgactctgaa gagctcctat agccttcaaa 780tgcacctaac tctactctaa aggcaaatgt cctcactggg aaatctgatc tgctgtttca 840gagaagtgca gggctacaca gtgtcttaca ctcctatcta ttgatgtttc ttggttttgc 900ctggtaatct gctgcttaaa tggattattt gatgacatat tgatattaaa acagtcctat 960<210> SEQ ID NO 20 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Repeatedunit in SEQ ID NO:1 <221> NAME/KEY: misc_feature <222> LOCATION:(3)..(3) <223> OTHER INFORMATION: Xaa is Asp or Ser <400> SEQUENCE: 20Gly Gly Xaa Phe Gly Gly 1 5 <210> SEQ ID NO 21 <211> LENGTH: 551 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 Met Ala GluGly Glu Asn Glu Val Arg Trp Asp Gly Leu Cys Ser Arg 1 5 10 15 Asp SerThr Thr Arg Glu Thr Ala Leu Glu Asn Ile Arg Gln Thr Ile 20 25 30 Leu ArgLys Thr Glu Tyr Leu Arg Ser Val Lys Glu Thr Pro His Arg 35 40 45 Pro SerAsp Gly Leu Ser Asn Thr Glu Ser Ser Asp Gly Leu Asn Lys 50 55 60 Leu LeuAla His Leu Leu Met Leu Ser Lys Arg Cys Pro Phe Lys Asp 65 70 75 80 ValArg Glu Lys Ser Glu Phe Ile Leu Lys Ser Ile Gln Glu Leu Gly 85 90 95 IleArg Ile Pro Arg Pro Leu Gly Gln Gly Pro Ser Arg Phe Ile Pro 100 105 110Glu Lys Glu Ile Leu Gln Val Gly Ser Glu Asp Ala Gln Met His Ala 115 120125 Leu Phe Ala Asp Ser Phe Ala Ala Leu Gly Arg Leu Asp Asn Ile Thr 130135 140 Leu Val Met Val Phe His Pro Gln Tyr Leu Glu Ser Phe Leu Lys Thr145 150 155 160 Gln His Tyr Leu Leu Gln Met Asp Gly Pro Leu Pro Leu HisTyr Arg 165 170 175 His Tyr Ile Gly Ile Met Ala Ala Ala Arg His Gln CysSer Tyr Leu 180 185 190 Val Asn Leu His Val Asn Asp Phe Leu His Val GlyGly Asp Pro Lys 195 200 205 Trp Leu Asn Gly Leu Glu Asn Ala Pro Gln LysLeu Gln Asn Leu Gly 210 215 220 Glu Leu Asn Lys Val Leu Ala His Arg ProTrp Leu Ile Thr Lys Glu 225 230 235 240 His Ile Glu Gly Leu Leu Lys AlaGlu Glu His Ser Trp Ser Leu Ala 245 250 255 Glu Leu Val His Ala Val ValLeu Leu Thr His Tyr His Ser Leu Ala 260 265 270 Ser Phe Thr Phe Gly CysGly Ile Ser Pro Glu Ile His Cys Asp Gly 275 280 285 Gly His Thr Phe ArgPro Pro Ser Val Ser Asn Tyr Cys Ile Cys Asp 290 295 300 Ile Thr Asn GlyAsn His Ser Val Asp Glu Met Pro Val Asn Ser Ala 305 310 315 320 Glu AsnVal Ser Val Ser Asp Ser Phe Phe Glu Val Glu Ala Leu Met 325 330 335 GluLys Met Arg Gln Leu Gln Glu Cys Arg Asp Glu Glu Glu Ala Ser 340 345 350Gln Glu Glu Met Ala Ser Arg Phe Glu Ile Glu Lys Arg Glu Ser Met 355 360365 Phe Val Phe Ser Ser Asp Asp Glu Glu Val Thr Pro Ala Arg Ala Val 370375 380 Ser Arg His Phe Glu Asp Thr Ser Tyr Gly Tyr Lys Asp Phe Ser Arg385 390 395 400 His Gly Met His Val Pro Thr Phe Arg Val Gln Asp Tyr GlnTrp Glu 405 410 415 Asp His Gly Tyr Ser Leu Val Asn Arg Leu Tyr Pro AspVal Gly Gln 420 425 430 Leu Ile Asp Glu Lys Phe His Ile Ala Tyr Asn LeuThr Tyr Asn Thr 435 440 445 Met Ala Met His Lys Asp Val Asp Thr Ser MetLeu Arg Arg Ala Ile 450 455 460 Trp Asn Tyr Ile His Cys Met Phe Gly IleArg Tyr Asp Asp Tyr Asp 465 470 475 480 Tyr Gly Glu Ile Asn Gln Leu LeuAsp Arg Ser Phe Lys Val Tyr Ile 485 490 495 Lys Thr Val Val Cys Thr ProGlu Lys Val Thr Lys Arg Met Tyr Asp 500 505 510 Ser Phe Trp Arg Gln PheLys His Ser Glu Lys Val His Val Asn Leu 515 520 525 Leu Leu Ile Glu AlaArg Met Gln Ala Glu Leu Leu Tyr Ala Leu Arg 530 535 540 Ala Ile Thr ArgTyr Met Thr 545 550

What is claimed is:
 1. A polynucleotide comprising the sequences of anyone of SEQ ID NOs: 1, 3, 5, 7, 9, 10, 12, 14, 16, 18, and
 19. 2. Anisolated polynucleotide having the sequence of (a) any one of SEQ IDNOs: 1, 3, 5, 7, 9, 10, 12, 14, 16, 18, and 19; (b) anaturally-occurring polynucleotide comprising a sequence of (a); or (c)a naturally-occurring polynucleotide having at least 70% identity with anaturally-occurring polynucleotide of (b) and, in naturally-occurringneural cells, has its expression modulated when the cells are subjectedto neurotoxic stress; (d) a naturally-occurring polynucleotide capableof hybridizing under moderately stringent conditions to anaturally-occurring polynucleotide of (b) and, in naturally-occurringneural cells, has its expression modulated when the cells are subjectedto neurotoxic stress; (e) a fragment of a polynucleotide of (a), (b),(c) or (d) having at least 20 nucleotides; or (f) a polynucleotidesequence complementary to a polynucleotide of (a), (b), (c), (d) or (e).3. An isolated polynucleotide in accordance with claim 2, wherein saidsequence of (a) is SEQ ID NO:
 3. 4. An isolated polynucleotide inaccordance with claim 2 comprising a strand of a full-length cDNA.
 5. Anisolated polynucleotide in accordance with claim 3 comprising a strandof a full-length cDNA.
 6. An isolated polypeptide comprising a proteinencoded by a cDNA in accordance with claim 4, a variant which has anamino acid sequence having at least 70% identity to said protein andretains the biological activity thereof, or a fragment of said proteinor variant which retains the biological activity thereof, or afunctional derivative or salt of said protein, variant or fragment. 7.An isolated polypeptide comprising a protein encoded by a cDNA inaccordance with claim 5, a variant which has an amino acid sequencehaving at least 70% identity to said protein and retains the biologicalactivity thereof, or a fragment of said protein or variant which retainsthe biological activity thereof, or a functional derivative or salt ofsaid protein, variant or fragment.
 8. An isolated polypeptide comprisingthe amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 11, 13,15, and 17, a variant which has an amino acid sequence having at least70% identity to said polypeptide and retains the biological activitythereof, or a fragment of said polypeptide or variant which retains thebiological activity thereof, or a functional derivative or salt of saidpolypeptide, variant or fragment.
 9. A molecule which comprises theantigen-binding portion of an antibody specific for a protein, variantor fragment in accordance with claim
 6. 10. A method of treating theeffects of stroke, hypoxia, and/or ischemia, comprising bringing intothe vicinity of the cells to be treated a polypeptide of SEQ ID NO: 4.11. A method for diagnosing cells which have been subjected to hypoxiaand/or ischemia, comprising assaying for RNA comprising a sequence ofany one of SEQ ID NOs: 1, 3, 5, 7, 9, 10, 12, 14, 16, 18, and 19 or forthe expression product of a gene in which one of said sequences is apart, the positive finding of said RNA or expression product indicatingthe likelihood that such cells have been subjected to hypoxia orischemia.
 12. In a method for screening drugs which up-regulate ordown-regulate a gene, the improvement wherein said gene is a gene whichis transcribed to an RNA containing a sequence in accordance with claim1.